Protocols for Characterization of Cdk5 Kinase Activity

Abstract

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This unit includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used with modification to measure the activity of other Cdks as well.

Basic protocol 1:

Measuring Cdk5 activity from protein lysates

Support protocol 1:

Immunoprecipitation of Cdk5 using Dynabeads

Alternate Protocol 1:

Non-radioactive protocols to measure Cdk5 kinase activity

Support protocol 2:

Western blot analysis for the detection of Cdk5, p35, and p39

Support protocol 3:

Immunodetection analysis for Cdk5, p35, and p39

Support protocol 4:

Genetically engineered mice (+ and − controls)

Basic protocol 2:

Identifying new Cdk5 substrates and kinase inhibitors

INTRODUCTION

Cyclin-dependent kinases (Cdks) are proline-directed serine/threonine kinases that play important roles in the control of cell division, but cyclin-dependent kinase 5 (Cdk5), on the other hand, differs from other Cdks as it is most active in postmitotic neurons. Originally, Cdk5 was identified and cloned from HeLa cells as a PSSALRE kinase with a 57% sequence homology to cdc2 (Meyerson et al., 1992). Cdk5 was additionally discovered and cloned from a wide variety of species including bovine brain (Lew et al., 1992b), rat brain (Hellmich et al., 1992), porcine brain (Hisanaga et al., 1995), Xenopus, and zebrafish (Gervasi and Szaro, 1995), all showing a remarkable 99% sequence identity with a high degree of conservation. Cdk5 is abundantly expressed in post-mitotic neurons, where its activity appears to primarily regulate neuronal processes rather than control cellular proliferation (Tsai et al., 1993). The kinase activity of Cdk5 mainly depends upon the binding to its activators p35 and p39, both of which possess a cyclin-box fold within their protein structure, although Cdk5 can also bind to and be activated by Cyclin I (Tarricone et al., 2001, Brinkkoetter et al. 2009). The Cdk5 regulatory subunits p35 and p39 share about 57% amino acid homology (Dhavan and Tsai, 2001), but Cdk5/p39 complexes tend to be less stable as compared to Cdk5/p35 complexes (Saito et al., 2013). While Cdk5 is expressed in many cell types and tissues, the neuronal-restricted expression of p35 and p39 essentially limits its kinase activity mostly to postmitotic neurons. However, newly described non-neuronal roles for Cdk5 have also been identified (Figure 1). The expression of p35 begins during early embryonic stages and peaks in neonates, with its expression generally highest in the cerebral cortex and hippocampus. In contrast, p39 expression appears postnatally and is mainly localized to the cerebellum (Prochazkova et al., 2017). The expression levels of the Cdk5 activators are the rate-limiting step for Cdk5 enzymatic activity, as the transgenic overexpression of Cdk5 in mice did not alter overall neuronal kinase activity but overexpression of the p35 resulted in increased kinase activity (Takahashi et al., 2005).

Figure 1.

Neuronal and non-neuronal functions of Cdk5. Cdk5 and its regulatory subunits p35 and p39 are primarily expressed in post-mitotic neurons. As such, Cdk5 has been primarily studied for its role in regulating neuronal activity, including synaptic plasticity, axonal guidance, neuronal migration, and pain signaling. Non-neuronal functions for Cdk5, however, have been recently identified including a role in angiogenesis, insulin secretion, and immune responses (Contreras-Vallejos et al., 2012; Sharma and Sicinski, 2020). With its activity mostly seen in post-mitotic neurons, a role for Cdk5 in cancer was often overlooked, unlike with other Cdks that are involved in the cell cycle but altered expression of Cdk5 and its activators has now been detected in several types of cancer. Pozo and Bibb, 2016) (generated through https://biorender.com/).

Although expressed in all tissues, Cdk5 is mostly known for controlling neuronal activity, particularly as its expression is highest in the nervous system (Dhavan and Tsai, 2001). The importance of Cdk5 in regulating neuronal development is seen where a loss-of-function mutation in humans causes lissencephaly and cerebellar hypoplasia (Magen et al., 2015). A role for Cdk5 in neurodevelopment was further validated by genetic deletion of Cdk5 in a mouse, which caused abnormal corticogenesis resulting in perinatal lethality (Ohshima et al., 1996). Overall, Cdk5 plays important developmental roles in neuronal migration, cortical layer formation, axon elongation, and dendrite arborization in many regions of the developing brain. Although genetic ablation of the Cdk5 activator p35 results in a significant loss of Cdk5 activity, p35−/− mice are still viable unlike Cdk5−/− mice (Chae et al., 1997, Ohshima et al., 2001). However, the p35−/− mice show cortical lamination defects analogous to the defects in the Cdk5−/− mice, which further illustrates a critical role for Cdk5 in neuronal migration and cortical layering. Deletion of the related subunit p39, however, causes no observable phenotypic defects, but null mice display impaired remyelination along with defects in axonal growth and dendritic spine formation (Ko et al., 2001, Bankston et al., 2013, Li et al., 2016, Ouyang et al., 2020). The embryonic lethality seen with genetic ablation of Cdk5 is essentially only recapitulated through compound deletion of both Cdk5 activators, p35 and p39, which suggests possible overlapping roles for these two regulatory subunits during neuronal development. Overall, p35-mediated induction of Cdk5 activity has been better studied than p39 because of the lack of a prominent phenotype and the instability of Cdk5/p39 complexes. As for Cyclin I, deletion of this cyclin activator causes no developmental defects, but Cdk5/Cyclin I activity may be important in reducing injury-induced apoptosis in post-mitotic cells such as neurons and kidney podocytes (Brinkkoetter 2009).

In contrast to the neurodevelopmental defects that are a result of decreased kinase activity, Cdk5 hyperactivity can also have pathological consequences, as increased Cdk5 activity has been linked with neurodegenerative diseases such as AD, PD and ALS (Kawauchi et al., 2014). Normally, Cdk5 activity is tightly regulated, but under conditions of neuronal stress, Cdk5 can become deregulated, leading to neuronal toxicity and cell death. Under neurotoxic conditions, aberrant Ca²⁺ influx can activate the protease calpain in the neuron, which can then cleave p35 into p25 and p10 (Kusakawa et al., 2000). p35 is generally tethered to the plasma membrane via myristylation of its N-terminal region, but cleavage of p35 by calpain results in mislocalization of Cdk5/p25 to the cytosol (Asada et al., 2008). In addition, p25 also forms a much more stable Cdk5/p25 hyperactive complex with a longer half-life than Cdk5/p35. The mislocalized Cdk5/p25 complex then typically proceeds to hyperphosphorylate cytoskeletal proteins like tau and the microtubule-associated protein MAP1B, both of which are found in neurofibrillary tangles (Kawauchi et al., 2005). While overexpression of p35 in a mouse can induce Cdk5 activity that, in turn, can affect behavior (Takahashi et al., 2005; Pareek et al., 2006), transgenic p35 mice do not have any observable neuronal morphological defects, but promoting Cdk5 hyperactivity with the overexpression of p25, in contrast, can lead to neurodegeneration and the formation of neurofibrillary tangles in the brains of mice (Cruz et al., 2003). So, in general, Cdk5/p35 appears to be essential for regulating neurophysiology, particularly during neuronal development, but Cdk5/p25 activity seems to be more associated with pathological events such as neurodegeneration (Cortés et al., 2019).

Numerous Cdk5 substrates have been identified, where most are involved in neuronal activity (www.phosphosite.org). Cdk5 mediated phosphorylation of a specific cellular substrate can depend on the subcellular localization of the active kinase complex. The regulatory subunit p35 normally localizes Cdk5 to the plasma membrane, yet Cdk5 has also been detected in the cell nucleus (Futatsugi et al., 2012). In contrast, cleaved p25 diverts Cdk5 to the cytosol, which can then readily phosphorylate cytoskeletal elements like Tau. As seen with cortical layering defects in the Cdk5−/− mice, Cdk5 activity has a key role in neuronal migration, where Cdk5 is known to affect actin dynamics by phosphorylating substrates such as Pak1, Ephexin1, and Neurabin-1 (Shah and Rossie 2018). The seizures detected in p35−/− mice also suggest a role for Cdk5/p35 in synaptic functions (Dhavan and Tsai, 2001). Cdk5 is reported to regulate synaptic plasticity along with memory formation and extinction. Cdk5 and p35 are enriched in synaptic membranes, where it is known to phosphorylate proteins involved in synaptic plasticity including Munc18, Synapsin1, Amphiphysin1, and Dynamin1 (Kawauchi, 2014). In the peripheral nervous system, Cdk5 has so far been shown to modulate pain perception. In particular, the Cdk5 activator p35 is expressed in both the dorsal root ganglia and trigeminal ganglia, primarily in small and medium diameter nociceptors (Pareek et al., 2006; Yang et al., 2007). Cdk5 can then phosphorylate key pain transducers such as TRPV1, TRPA1, and P2X2a along with the cytoskeletal regulatory protein CRMP2, which modulates Cav2.2 and Nav1.7 activity (Pareek et al., 2007; Hall et al., 2018; Coddou et al., 2017; Gomez et al., 2020).

Because Cdk5 is primarily expressed in neurons, non-neuronal roles for Cdk5 activity were initially overlooked, but numerous studies have now begun to link Cdk5 activity with physiological roles outside the nervous system including cancer (Figure 1) (Contreras-Vallejos et al., 2012; Sharma and Sicinski, 2020; Pozo and Bibb, 2016). For example, Cdk5 is thought to influence inflammatory responses, particularly in controlling T-cell activation (Pareek et al., 2010). Cdk5 activity is also thought to be involved in glucose transport within adipocytes and to affect insulin secretion (Contreras-Vallejos et al., 2012), both of which may have implications in diabetes. Lastly, Cdk5 activity has been linked with angiogenesis, myogenesis, and DNA damage responses (Sharma and Sicinski, 2020). Although Cdk5 shares about 60% homology with Cdk1 and Ckd2, Cdk5 was essentially not studied in terms of cancer at first because its activity was thought to be mainly limited to post-mitotic neurons. Increased expression of Cdk5, p35, and p39, however, has recently been reported in several tumors (Pozo and Bibb, 2016). In terms of the cell cycle, Cdk5 was surprisingly found to phosphorylate Rb, which then can lead to increased E2F transcription factor activity (Futatsugi et al., 2012). Cdk5 is also suspected of influencing cancer cell migration, particularly because of its established role in modulating neuronal migration (Contreras-Vallejos et al., 2012).

In conclusion, Cdk5 is a key neuronal kinase that is implicated in both neuronal development and neurodegeneration while having identified roles outside the nervous system as well. Novel functions are continuously being identified for Cdk5 since its first discovery in 1992. A means of detecting kinase enzymatic activity is therefore essential in terms of identifying new roles for Cdk5 in both neuronal and non-neuronal cellular activities. This protocol chapter details methods for detecting Cdk5 along with its regulatory subunits and for monitoring its kinase activity in cultured cells and tissues (Figure 2). We provide techniques for identifying new Cdk5 substrates, testing new Cdk5 inhibitors, and evaluating Cdk5 activity in protein lysates. Some of the techniques detailed below can also be applied to other kinases as a basic method for tracking kinase activity.

Figure 2.

Depiction of Cdk5 radioactive assay. A) Structure of the Cdk5/p25 complex (van der Waals surface map of PDB3O0G, generated through https://biorender.com/). B) Substrate sequence site for Cdk5 (Details about Cdk5 can be found at https://www.phosphosite.org/, including a list of substrates). Cdk5 is a proline-directed serine/threonine kinase with an (S/T)P phosphorylation site, but half of the Cdk5 substrates typically contain an (S/T)PX(K/H/R) motif with a basic residue in amino acid position +3 (Borquez et al., 2013). C) In the Cdk5 assay, Cdk5 transfers a phosphate group labeled with ³²P onto a preferred substrate. The degree of kinase activity (via transfer of the radioactively labeled phosphate group) is then measured either using D) a scintillation counter or E) through SDS-PAGE followed by the detection of radioactivity with autoradiography film.

BASIC PROTOCOL 1:

MEASURING CDK5 ACTIVITY USING PROTEIN LYSATES

Detecting Cdk5 activity in tissues and cell culture has been useful for understanding its role in neurotoxicity, pain, cancer progression, etc. A means of measuring kinase activity is also important for discovering new functions for Cdk5 in various cellular activities and for following aberrant Cdk5 hyperactivity in pathological conditions. This protocol will detail a method for immunoprecipitating Cdk5 from cultured cells or tissues for subsequent use in a kinase assay. Essentially, Cdk5 is immunoprecipitated from a protein lysate using an anti-Cdk5 antibody and Protein A/G beads. Some of the captured Cdk5 will still be complexed to either p35 or p39 (or the calpain cleaved p25 or p29). Phosphorylation of Cdk5 itself at either Tyr15 or Ser159 may also possibly play a role in regulating Cdk5 activity (Shah and Lahiri 2014), although phosphorylation of Tyr15 appears to only occur on inactive monomeric Cdk5 (Kobayashi et al. 2014). Through immunoprecipitation, Cdk5 can be isolated in a given sample and its kinase activity measured, where the expression level of its regulatory subunits generally governs the degree of kinase activity.

Materials

Protein Lysis:

• Protein Lysis Buffer (see recipe in Reagents and Solutions):

  • T-PER (Thermofisher Scientific #78510)
  • Complete mini protease (Roche #04 906 837 001)
  • PhosSTOP™, Phosphatase inhibitor tablet (Roche #04 906 837 001)

• KIMBLE® DUALL® Tissue Grinder (1 or 5 ml) – for tissue homogenization or

• Cell scraper (Sarstedt #83.1832 or 83.1830) – for cultured cells

• Pierce™ BCA Protein Assay kit (Thermofisher Scientific #23227)

Immunoprecipitation:

• Protein A/G PLUS-Agarose, 25% slurry in PBS (Santa Cruz Biotechnology #sc-2003)

• Cdk5 antibody (AA 1–292) (antibodies-online Inc #ABIN514281 - unpurified serum, concentration of antibody can vary by lot) or anti-Cdk5 antibody (My Biosource #MBS9131167 - purified serum, concentration of antibody can vary by lot).

• Note: The Cdk5 Antibody (C-8) (SantaCruz Biotechnology #sc-173) was previously used in numerous publications to immunoprecipitate Cdk5, but this product has been discontinued.

• Control IgG Antibody: AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Immunoresearch #111–005-003) or AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson Immunoresearch # 115-005-003)

Cdk5 Kinase Reaction:

• Kinase Buffer 5X (KB5X) (see recipe in Reagents and Solutions):

  • MOPS Buffer (0.5 M, pH 7.4) (Boston Bioproducts #BB-2192)
  • Beta GlyceroPhosphate Solution (0.2 M, Serine and Threonine Phosphatases Inhibitor) (Boston Bioproducts #BP-435)
  • EGTA (0.5 M, pH 7.4, Autoclaved) (Boston Bioproducts #BM-721)
  • EDTA (0.5 M, pH 7.4, Autoclaved) (Boston Bioproducts #BM-711)
  • 2M MgCl₂ (Quality Biological Inc #340–034-721EA)
• Histone Mix (see recipe in Reagents and Solutions):

  • Cdk5 Substrate - Histone H1 (1 mg/ml) (Millipore Sigma #H5505–25mg)
• ATP cocktail (see recipe in Reagents and Solutions):

  • DTT 1 M (Millipore Sigma #3483–12-3)
  • 100 mM ATP cold (Millipore Sigma #A2383–25G)
  • Halt Protease/Phosphatase inhibitor cocktail (Thermofisher Scientific #78441)
  • ATP, [γ−³²P] unpurified (PerkinElmer NEG035C001MC)

• Cdk5/p35: active, GST tagged human (Millipore Sigma #SRP5011–10μg) or

• Cdk5/p25: active, GST tagged human (Millipore Sigma # C0745–10μg)

SDS-PAGE:

• NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.5 mm, 10-well (Separation Range: 3.5 to 260 kDa) (Thermofisher Scientific #NP0335BOX)

• NuPAGE™ MOPS SDS Running Buffer (20X) (Thermofisher Scientific # NP0001)

• SeeBlue™ Plus2 Pre-stained Protein Standard (Thermofisher Scientific # LC5925)

• XCell SureLock™ Mini-Cell (Thermofisher Scientific # EI0001)

• Laemmli (6X, SDS-Sample Buffer, Reducing) – (Boston BioProducts #BP- 111R)

• PowerPac™ Basic Power Supply (Bio-Rad #1645050) or equivalent

Coomassie Blue Stain and Destaining Solution (see recipe in Reagents and Solutions):

• Coomassie blue (Boston BioProducts #ST-120)

• Methanol (HPLC), (Fisher Chemical Fisher Scientific #A452–4)

• Acetic acid glacial (VWR #V193–14)

Radioactivity (working with ATP [γ−³²P]):

• follow your institute’s guidelines for the proper use and handling of radioactive material

• Nalgene™ Acrylic Benchtop Beta Radiation Shield (Thermofisher Scientific # 6700–1812 or 6700–2418) or equivalent

• 5 Gallon Carboy with Handles (Research Product International #2210–0050) or equivalent

• Funnel

• Rad-3 Lock Box, 5 Gallon Carboy Capacity (Research Product International #RLB-03) or equivalent

• Model 3 General Purpose Survey Meter (Ludlum Measurements, Inc #48–1605) or equivalent

• Model 44–9 Alpha-Beta-Gamma Detector (Ludlum Measurements, Inc #47–1539) or equivalent

• Defenders® 5 Gallon Step Can (Rubbermaid Commercial Products # FGST5EGLRD) or equivalent

• Rad-4 Lock Box, Large Capacity Unit (Research Product International #RLB-04) or equivalent

• Corrugated Waste Box for Radioactive Material (Research Product International #BRS-15) or equivalent

• Heavy Duty Poly Liners (Research Product International #BRS-16) or equivalent

• Caution Radioactive Material Tape (Research Product International #140046) or equivalent

• COUNT-OFF Surface Cleaner (PerkinElmer #6NE942T) or equivalent

Lab Equipment:

• Thermomixer™ R (Eppendorf™ #05–400-205) or equivalent

• Norlake Freezer −20 (Thomas Scientific #1156Y04) or equivalent

• HB-500 Minidizer Hybridization Oven (Lab Rep Co #95–0330-01) or equivalent

• Centrifuge (Eppendorf™ #5415C) or equivalent

• Refrigerated Micro Centrifuge (Eppendorf™ #5417R) or equivalent

• IBI Scientific™The Belly Dancer™Orbital Platform Shaker (Fisher Scientific

• #15–453-211) or equivalent

• Mini Vortex-Genie 2 (Daigger #G22220) or equivalent

• Sample mixer for rotation of tubes (e.g., HulaMixer™ Sample Mixer - Thermo Scientific #15920D)

• Imager (Fluorochem M System Bio-Techne #92–15312-00) or equivalent gel scanner

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• Fisherbrand™ MBP™ Wide Bore Pipette Tips (Fisher Scientific #02–707-600)

• For mixing and pipetting agarose beads

• MμltiFlex™ Pipet Tips (VWR #53550–167)

• For gel loading

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tube with lock lid (Crystalgen #23–2052LK)

• For overnight immunoprecipitation

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• 2.0 ml Microcentrifuge Tube (Stellar Scientific #T20–100) or equivalent

• Nalgene™ Microcentrifuge Tube Rack (Thermofisher Scientific #5973–0015) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Utility Boxes (Thermofisher Scientific #57000500) or equivalent

• Ice Bucket

• Gel Knife (Thermofisher Scientific #EI9010)

• Heavy-Duty Long-Blade Scissors (Fisher Scientific #14–275C)

• Filter forceps, blunt end (Millipore Sigma #XX6200006P)

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

Film Development:

• HyBlot CL® Autoradiography Film (Denville Scientific #E3018) or equivalent

• Film Cassette (Amersham Hypercassette Autoradiography Cassettes #RPN11649)

• Transparency (3M Highland 901 8.5 in × 11- inch Transparency Film)

• Dark Room and Developer (Medical Film Processor # SRX-101A)

Preparing Protein Lysates

Tissue or cell lysates were prepared using in T-PER Buffer plus protease and phosphatase inhibitors (see protein lysis buffer in Reagents and Solutions). T-PER is a proprietary detergent in 25 mM bicine and 150 mM sodium chloride, but high concentrations of detergent reportedly can affect Cdk5/p39 activity, so a different lysis buffer composition may be required to study p39 (see Bankston et al., 2017):

Tissues from experimental mice should be harvested shortly after euthanasia, then freeze on dry ice to reduce post-mortem generation of p25 (store frozen tissues at −80°C). Often, a 1:10 ratio (100 mg of tissue / 1.0 ml of lysis buffer) is used for homogenization, but the proportions of lysis buffer to tissue may need to be adjusted depending on the expression of Cdk5 and its activators. Typically, we homogenize tissues using a KIMBLE® DUALL® Tissue Grinder (1 or 5 ml) on ice, but other means of homogenization are acceptable, such as the Precellys homogenizer supplied by Bertin instruments.

As controls, one may want to include tissues from p35−/− mice (with significantly reduced Cdk5 activity [Chae et al., 1997, Ohshima et al., 2001]) and/or Tgp35 mice (~50% increase in Cdk5 activity [Takahashi et al., 2005, Prochazkova et al., 2013]) along with their wild type littermates.

For tissue culture, add enough cold T-PER to cover the cell surface area. Then use a cell scraper to harvest the cell lysate.

Protein lysates are transferred into a microfuge tube, left on ice for 30 minutes, vortexed, then centrifuged at maximum speed for 30 minutes. Collect the supernatant and store the lysates at −80°C. RNA or DNA can be retrieved from the remaining pellet using the appropriate nucleic acid extraction method (Store protein lysates at −80°C).

Measure protein concentration with Pierce™ BCA Protein Assay kit or equivalent assay according to the manufacturer’s protocol.

Protein A/G Agarose Antibody Conjugates

An immunoprecipitation step is first needed to pull down Cdk5 from the protein lysates. Facets of immunoprecipitation are detailed in a Current Protocols in Cell Biology chapter by Bonifacino et al., 2016. (Unit 7.2). Protein A/G Agarose; Antibody conjugates are prepared before overnight immunoprecipitation with the test lysates. Generation of Protein A/G Agarose-Antibody conjugates can be performed at room temperature.

Each sample requires 50 μl of Protein A/G Agarose. If working with a small number of samples, the agarose beads can be washed individually (detailed below), and the measured sample protein lysates (see Step 6 below) are added to the Protein A/G Agarose-Antibody conjugates following the final wash. Special care must be performed during the washes so as not to lose any of the agarose beads (see Troubleshooting below). We prefer to make a bulk prep of Protein A/G Agarose;Antibody conjugates to simplify washing. The following is for ~ 8 samples (suitable for a 10-well gel plus control and the protein ladder). Can be scaled appropriately

• 1Resuspend Protein A/G Agarose beads (Vortex briefly and mix by pipetting). Take 500 μl of the Protein A/G Agarose bead slurry (~50 μl/sample: for 8 samples + 2 extra = 500 μl) from the bottom of the tube and transfer into a 2ml tube.
• 2Spin the Protein A/G Agarose beads at 6,000 × g for 60 sec.
• 3Wash Step (2X): Carefully aspirate the supernatant and discard. Resuspend agarose beads with 1000 μl 1X TBS. Mix by inverting a couple of times. Centrifuge 6,000 × g for 60 sec. Repeat one more time.
• 4Resuspend with 1000 μl 1X TBS.
• 5Add 50 μl anti-Cdk5 Ab (~ 5 μl /sample) - Do not vortex. Place the tube on a rotator and incubate at room temperature for 2 hours to generate Protein A/G Agarose-Antibody conjugates. If enough available protein, a negative control can be made using a control IgG (use 2 μg/sample) instead of the anti-Cdk5 antibody.

While the Protein A/G Agarose;Antibody is mixing, one can prepare the protein samples for immunoprecipitation.

• 6Measure out 200 μg of protein for each sample in a microfuge tube (use a tube with a locking cap). Can go as low as 50 μg for neuronal tissue where Cdk5 is highly expressed, but may need more protein, between 200–500 μg, when working with cultured cells. Then add lysis buffer to bring the total volume up to 500 μl with the protein lysis buffer. Keep on ice until needed.

Immunoprecipitation of Cdk5

• 7Spin the Protein A/G Agarose-Antibody mixture (step 5) at 6,000 × g for 60 sec.
• 8Wash Step (3X): Carefully aspirate the supernatant and discard. Resuspend agarose beads with 1000 μl 1X TBS. Mix by inverting a couple of times. Centrifuge 6,000 × g for 60 sec. Repeat the wash step two more times.
• 9Resuspend agarose beads in 1000 μl 1X TBS or protein lysis buffer. Mix well.
• 10Add 100 μl of agarose beads slurry to each protein sample (from step 6).
• 11Mix on a rotator in a cold room or 4°C refrigerator overnight.

While preparing the Protein A/G Agarose-Antibody mixture, an additional prep of Protein A/G Agarose beads can be washed then refrigerated at 4°C for later use. Washed Protein A/G Agarose beads without antibodies will be needed to make the negative control and, if feasible, for a positive control using either recombinant Cdk5/p25 or Cdk/p35.

Cdk5 Kinase Assay

• 12Spin agarose beads (Cdk5 captured Protein A/G Agarose-Antibody mixture) at 6,000 × g for 60 sec. Visually inspect beads to ensure equal pipetting of the Protein A/G agarose slurry.
• 13Wash Step (3X): Carefully aspirate the supernatant. The supernatant should be frozen and stored in order to examine the efficiency of immunoprecipitation if unexpected results are obtained. Resuspend agarose beads with 1000 μl 1X TBS (One can alternatively use the protein lysis buffer for washing). Mix by inverting a couple of times. Centrifuge 6,000 × g for 60 sec. Use a refrigerated microfuge set at 4°C and keep samples on ice.
• 14Perform a final wash by resuspending agarose beads in 250 μl of 1X Kinase buffer (see recipe in Reagents and Solutions). Spin 600 × g for 60 sec. at 4°C. Resuspend agarose beads in 100 μl of 1X Kinase buffer.
• 15Mix beads thoroughly by pipetting up and down. Use wide-bore pipette tips. Take 20 μl of the above agarose bead concoction into a new labeled microfuge tube. If feasible, run a duplicate sample. Refrigerate the remaining beads for future use if needed.
• 16The negative control will just be 20 μl of the washed bead slurry. For positive control, dilute recombinant Cdk5/p25 (or p35) 1:250 (1:250 is a starting dilution of the recombinant active kinase used to obtain a definitive radioactive signal but a higher dilution of the enzyme may be desired – see Figure 3 and 5). Add 5 μl enzyme to 15 μl of the washed beads.
• 17Prepare the Histone H1 Mixture (10 μl KB5X and 10 μl Histone H1 - see recipe in Reagents and Solutions) and add a total of 20 μl / sample.
• 18Prepare the cold ATP cocktail (see recipe in Reagents and Solutions). Dispense 200 μl into a new tube as a working solution.

Figure 3.

Cdk5 activity was measured in wild-type (WT) and transgenic p35 overexpressing (Tgp35) mice. Protein A/G Agarose-Antibody conjugates were generated using either a control IgG antibody or with anti-Cdk5 antibody. Results show that Tgp35 mice have more Cdk5 activity in the brain compared to WT mice due to overexpression of the Cdk5 activator p35. A positive control (+Con) with either recombinant Cdk5/p35 or Cdk5/p25 was included to ensure that the experimental reagents and conditions are working properly (a 1:250 dilution was run but a higher dilution of the recombinant active kinase may be needed).

Figure 5.

Cdk5 was immunoprecipitated from wild type (WT) and transgenic p35 overexpressing (Tgp35) mice using Dynabeads™. The Cdk5 activity results from the brain lysates are similar to Figure 3, where Cdk5 was pulled down using Protein A/G Agarose beads instead. Again, the Tgp35 display more Cdk5 activity than the control wild-type littermate (+Con = positive control of recombinant kinase).

All procedures from this step onwards should be performed in a designated radioactive area using the proper plexiglass shielding. All waste solutions along with dry waste materials (used gloves, pipet tips, etc.) should be disposed of properly in the designated radioactive waste containers according to your institute’s radioactivity protocols.

• 19Add 10–20 μl of hot ATP ([γ−³²P) (see Reagents and Solutions) into 200 μl of ATP cocktail (100–200 μCi)
• 20Add 10 μl of this hot ATP mixture to the samples (total volume 50 μl). Mix thoroughly by gently tapping the microfuge tube (No vortexing) and incubate at 30°C for 1 hour using an incubator (such as a Mini Hybridization Oven).

SDS-PAGE

We use NuPAGE™ gels – be sure to assemble the XCell SureLock™ Mini-Cell as described in the NuPAGE™ gel manual.

• 21Stop the kinase reaction by adding 10 μl of Laemmli (6X SDS) sample buffer. Heat at 95°C for 5 minutes in a Thermomixer™, then let the samples cool to R.T.
• 22Spin down. Load 25–35 μl of the kinase reaction (do not pipette up the beads) into a well of a NuPAGE™ 4–12% Bis-Tris Protein Gels. Be sure to include the protein ladder (10 μl SeeBlue™ Plus2 marker). Set up separate gel if you prepared duplicate samples.
• 23Run gel electrophoresis at 100 volts for 1 ½ hour.

Coomassie blue staining

Coomassie blue staining of the gel is needed to visualize Histone H1. All steps are done at room temperature using an orbital shaker. Dispose of all finished solutions in an appropriate radioactive waste container.

• 24Release the gel from its plastic cassette using a gel knife. Wash the gel with distilled water twice, 5 mins per wash on the rotator. Can use the lid of a pipette tip box or small tray.
• 25Stain gel with Coomassie blue. Use enough stain to cover the gel. Incubate 1–2 hours.
• 26Destain overnight at R.T. Cover the top and bottom of the gel with a Kimwipe, then add the destaining solution (see Reagents and Solutions). For destaining, use a tray that can be covered with a lid for overnight incubation.
• 27Then wash 3 times with water, 5 mins per wash.

Detection of Radioactivity

Radioactive labeling of Histone H1 is detected using autoradiography film. This requires an available darkroom and a functioning developer. Do not expose the film to light until developed.

• 28Cut two sheets of transparency film to fit inside the film cassette. Tape one onto the bottom of the cassette.
• 29Place gel into film cassette on top of the transparency. May need to cut off the raised foot of the gel make level. Cover with a 2^nd transparency and tape down.
• 30Take the film cassette to a dark room and add a piece of autoradiography film onto the gel. Expose the film at different time intervals to get the best bands.
• 31A picture or scan of the Coomassie-stained gel will eventually need to be taken after adequate exposure of the bands is achieved.

Generally, we expose the film overnight, depending on the signal from the Geiger counter. If the signal is very intense, we then develop the film for about 2 – 4 hrs. The film can be developed at room temperature (Figure 3).

SUPPORT PROTOCOL 1:

IMMUNOPRECIPITATION OF CDK5 USING DYNABEADS

Due to their ease of use, researchers have been switching to magnetic beads for immunoprecipitation (see Troubleshooting below). Magnetic separation of the beads helps to prevent sample loss that can occur by pipetting errors throughout the multiple wash steps. Magnetic beads can also save time by eliminating the need for centrifugation, which tends to disrupt the immune complexes.

To immunoprecipitate with magnetic beads, we suggest the following protocol:

Materials

Dynabeads™ Protein G (Thermo Scientific #10003D) (will need DynaMag™−2 Magnet for Cdk5 immunoprecipitation - Thermo Scientific #12321D)

Antibodies (Figure 4)

Figure 4.

Cdk5 assay was performed using Dynabeads™ with anti-Cdk5 antibody (~2 μg) from different companies. Cdk5 was immunoprecipitated from wild-type mouse brains. Lane 1 is C-8 (SantaCruz Biotechnology #sc-173), which has now been discontinued. The commercially available antibodies in Lanes 2 and 3 were not efficient in pulling down Cdk5. The best activity was achieved with antibody from antibodies-online Inc. (Lane 4) and Mybiosource (Lane 5). Lanes 6 and 7 are negative controls with mouse and rabbit IgG, respectively. The kinase reaction was terminated by spotting onto P81 phosphocellulose filter paper, where the level of radioactivity was later measured using a scintillation counter (see Basic Protocol 2 below). Radioactivity recorded in CPM (counts per minute) (Amin et al., unpublished data).

• Cdk5 antibody (AA 1–292) (antibodies-online Inc #ABIN514281) or anti-Cdk5 antibody (My Biosource #MBS9131167).

• Control IgG Antibody: AffiniPure Goat Anti-Rabbit or Anti-Mouse IgG (H+L) (Jackson Immunoresearch #111-005-003)

Wash Buffer

• PBS pH 7.4 with or without 0.02% Tween™ 20 (Millipore Sigma #P9416)

Sample mixer for rotation of tubes (e.g., HulaMixer™ Sample Mixer - Thermo Scientific #15920D)

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tube with lock lid (Crystalgen #23–2052LK)

• For overnight immunoprecipitation

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• Nalgene™ Microcentrifuge Tube Rack (Thermofisher Scientific #5973–0015) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Ice Bucket

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

Follow the user manual to immunoprecipitate using Dynabeads™ (See thermofisher.com/magnets for recommendations):

1. Per sample, pipet 50 μl of the Dynabeads™ into a microfuge tube. Be sure the Dynabeads™ are in solution by vortexing before pipetting. Prepare a tube for Positive and Negative Control.

2. Place on the magnetic rack to separate the beads from the suspension. Discard the supernatant then remove the tubes from the magnet. Resuspend the beads in 500 μl of the wash buffer (PBS with 0.02% Tween™ 20)

3. Add 10 μl of Cdk5 antibody (antibodies-online Inc). For the Negative Control, add 2μg of a control IgG antibody. Nothing needs to be added to the Positive Control (recombinant Cdk5 is added later).

4. Incubate 30 minutes at room temperature on the mixer.

5. After incubation, place the tubes on the magnetic rack and remove the supernatant.

6. Wash Step (1X): Remove the tubes from the magnet and resuspend the Dynabeads™ with 500 μl of the wash buffer. Mix thoroughly by pipetting. Do not invert. Then, place the tubes on the magnetic rack and, after the beads are separated from the solution, remove the supernatant.

7. Resuspend in 500 μl of the prepared protein samples (~200 μg of protein in a volume of total a of 500 μl of the protein lysis buffer – see Step 6 of Basic Protocol 1 and Reagents and Solutions for the lysis buffer formulation). For the Positive and Negative Control, just resuspend in 500 μl of the protein lysis buffer.

8. Incubate for 1 hour at room temperature on the mixer.

9. After incubation, place the tubes on the magnetic rack and remove the supernatant.

10. Wash Step (3X): Remove the tubes from the magnet and resuspend the Dynabeads™ with 500 μl of the wash buffer. Mix thoroughly by pipetting. Then, place the tubes on the magnetic rack and, after the beads are separated from the solution, remove the supernatant. Repeat two more times.

11. Wash Step (1X): Remove the tubes from the magnet and resuspend the Dynabeads™ with 250 μl of the 1X kinase buffer (see Reagents and Solutions). Mix thoroughly by pipetting. Then, place the tubes on the magnetic rack and, after the beads are separated from the solution, remove the supernatant.

12. Resuspend the Dynabeads™ with 100 μl of 1X kinase buffer.

13. Mix beads thoroughly by pipetting up and down (~5 times or until the Dynabeads™are in solution). Take 20 μl of the above bead concoction into a new labeled microfuge tube. If feasible, run a duplicate sample. Refrigerate the remaining beads for future use if needed.

14. For the Positive Control, take 15 μl of the washed beads and add 5 μl of diluted recombinant Cdk5/p35 or Cdk5/p25 (start with a 1:250 dilution in 1X kinase buffer but higher dilution may be preferred).

15. Follow Step 17 of Basic Protocol 1 onwards for the remainder of the Cdk5 assay.

Essentially, add 20 μl of the Histone H1 Mixture (10 μl KB5X and 10 μl Histone H1 - see recipe in Reagents and Solutions). In a designated radioactivity room, add 10 μl of the hot ATP mixture (see Reagents and Solutions). Incubate for 1 hour at 30°C. Stop the kinase reaction by adding 10 μl of Laemmli (6X SDS) sample buffer. Heat at 95°C for 5 minutes, then let the samples cool. Run phosphorylated Histone H1 on NuPAGE™ 4–12% Bis-Tris Protein Gels (Figure 5)

ALTERNATE PROTOCOL

NON-RADIOACTIVE PROTOCOLS TO MEASURE CDK5 KINASE ACTIVITY

Kinase activity can be measured using different assay formats including electrochemical assays (Shin et al., 2014; Zhao et al., 2015), mass spectrometry (Kim et al., 2007), colorimetric assays (Wang et al., 2006; Wei et al., 2008; Zhou et al., 2014), immunoassays (Farley et al., 1992, Schraag et al., 1993) in addition to radiolabels (Hutti et al., 2004). Different factors need to be considered to choose the correct assay format to get the optimum results. High sensitivity and specificity are needed. Nowadays, different kinds of readout technologies are available for Cdk5 kinase measurements. Cdk5 uses ATP, Mg²⁺, and a peptide/protein substrate during the kinase reaction. Some protocols detect the phosphorylation of substrate to generate a phosphopeptide/phosphoprotein while other methods detect the disappearance of ATP or the formation of ADP. Using [γ−³²P] ATP is a sensitive and accurate means of measuring Cdk5 activity, but the problems associated with this method are the cost involved in the safe disposal of radioactive material, mandatory requirements for safe handling of radioactive reagents, and licenses to get the permission for use of radioactivity. Moreover, the user will need special training to use radioactive material.

The most basic non-radioactive method for measuring Cdk5 activity is with a phospho-Histone-H1 antibody (i.e. anti-histone H1 phosphorylated - Millipore Sigma #06–597) that specifically detects phosphate addition onto a known Cdk Histone-H1 phosphorylation motif (Piedrahita et al., 2010). This method is not adaptable for use in identifying new substrates, however, so an alternative to this strategy is with the use of an anti-thiophosphate ester antibody. This kinase assay uses ATP-γ-S (available through Abcam #ab138911) (Zhuang et al., 2018). p-Nitrobenzyl mesylate (#ab138910) is used to alkylate the thiophospholyation site on the substrate, which is then detected by a thiophosphate ester rabbit monoclonal antibody (#ab92570) using Western blot. Fluorescent detection of Cdk5-mediated phosphorylation has also been reported as a means for measuring kinase activity. With Cdk5-Act (Peyressatre et al., 2020), a fluorescently labeled peptide substrate is linked to a phosphoamino acid-binding domain (PAABD) that subsequently prevents progressive quenching of the fluorescence after the substrate is phosphorylated. The Z’-LYTE assay, in contrast, uses Fluorescence Resonance Energy Transfer (FRET) to detect Cdk5 activity. With the Ź-LYTE™ kinase assay kit–Ser/Thr 12 peptide from ThermoFisher Scientific #PV3673, Cdk5 mediated phosphorylation of a synthetic FRET-peptide later prevents cleavage by a site-specific protease. The uncleaved peptide maintains FRET between coumarin and fluorescein fluorophores that would be disrupted with a cleaved peptide.

The depletion of ATP to ADP that occurs during the kinase reaction can alternatively be used to quantify kinase activity instead of following the direct phosphorylation of a substrate by Cdk5 with the techniques mentioned above. The Cdk5/p35 ADP-Glo™ Kinase Assay (Promega ADP-Glo™ Kinase Assay #V9101, Cdk5/p35 Kinase Enzyme System #V3271, ADP-Glo™ + Cdk5/p35 Kinase Enzyme System #V9551) determines Cdk5 activity by measuring the accumulation of ADP during the kinase reaction via a luciferase reaction. The ADP-Glo™ Kinase Assay has recently been applied to finding new non-neuronal functions of Cdk5 in both immune responses and endothelial cell migration (Lam et al., 2015; Na et al., 2015; Lampropoulou et al., 2018). The ADP-Glo™ Kinase Assay can be performed in either a 96 or 384 multiwell plate and uses a simple two-step reaction to tract Cdk5 activity. Following the kinase reaction, an equal amount of ADP-Glo™ Reagent is added to each sample well to terminate the reaction and deplete any residual unused ATP. After a 40 minute incubation, the Kinase Detection Reagent is next added to convert ADP to ATP that then fuels a luciferase/luciferin reaction. Within 30–60 minutes, the multiwell plate can then be read on a luminometer, where the amount of ADP that was generated during the kinase reaction is ultimately written as relative light units (RLU) (Figure 6). A white multiwell plate is needed for detecting the luciferase reaction where a 25μl kinase reaction volume is suggested when using a 96 well plate. 25μl of the ADP-Glo™ Reagent is then added to each sample well, followed by 50μl of Kinase Detection Reagent. For a 384 multiwell plate, the kinase reaction can be minimized to a 5μl kinase reaction volume with the ADP-Glo™ reagents added in similar 1:1:2 proportions. With the ability to be scaled down to a 384 well format, a key application of the ADP-Glo™ Kinase Assay is to test kinase inhibitors using recombinant Cdk5 (see the ADP-Glo™ application notes on Cdk5/p35 by Zegzouti et al.; Sanphui et al., 2013). Measuring Cdk5 activity in cultured cells or from tissue samples, however, requires immunoprecipitation of Cdk5 using Protein A/G Agarose beads. We could not obtain consistent results with protein lysates, so immunoprecipitation is needed, where the agarose beads used to pull down the Cdk5/p35 are added directly to the kinase assay. The agarose beads, however, give a high background using the luminometer, which needs to be subtracted from the acquired readings. The overall kinase reaction calls for Cdk5, buffer, ATP, and a designated substrate to be incubated for 30–60 minutes at 30°C. Histone H1 is commonly used as a Cdk5 substrate, but Histone H1 can be phosphorylated by other kinases as well. An alternate substrate is to use an NF-H (neurofilament H - VKSPAKEKAKSPVK) peptide instead (a small peptide can be used with this assay since the phosphorylated end product is not run on a gel). NF-H is specifically found in nerve bundles and contains a KSPX Cdk5 phosphorylation motif that makes it an ideal target to measure Cdk5 activity. The ADP-Glo™ Kinase Assay can also be useful for identifying new Cdk5 substrates too, such as mSin3a, which is a component of the HDAC1 repressor complex (Lam et al., 2015).

Figure 6.

Specificity and linearity of the ADPGlo Kinase Assay. Graphs A and B depict increasing concentrations of the Cdk5/p25 and the corresponding luminescence associated with the activity of the Cdk5 kinase. For graphs C and D, Cdk5 was immunoprecipitated from the wild type and knockout brains then tested for kinase activity. The immunoprecipitation of Cdk5 from the Cdk5 and p35 wild type and knockout brain samples show increased Cdk5 activity in the control wild type samples versus the knockout samples.

SUPPORT PROTOCOL 2:

WESTERN BLOT ANALYSIS FOR THE DETECTION OF CDK5, P35, AND P39

In addition to measuring Cdk5 activity using the above kinase assay, the protein expression of Cdk5 and its activators, p35 and p39, should be assessed by Western blot. Overall Cdk5 activity is tightly controlled by the amount of p35 and p39 expression and, to some degree, by regulatory phosphorylation at Thr14, Tyr15, and Ser159 (shared phosphorylation sites in common to other Cdk’s) (Shah and Lahiri, 2014). The binding of Cdk5 with its activators p35, p39, and Cyclin I, however, appear to inhibit phosphorylation of Cdk5 on Tyr15, which is only detected on monomeric inactive Cdk5 (Kobayashi et al. 2014). As mentioned, our lab has determined that the expression levels of p35 in particular is a key rate-limiting step for Cdk5 enzymatic activity (Takahashi et al., 2005). It can therefore be useful to pair the results of the radioactive kinase assay with Western blot measuring the expression of p35 to fully understand the kinetics of Cdk5 activity in a given sample. In addition, conversion of p35 to the calpain cleavage product p25 should be investigated to fully interpret the Cdk5 kinase assay results and to evaluate the possible occurrence of neuro-toxicity or neurodegeneration. For Western blot, we use the NuPAGE™ gels according to the manufacturer’s protocol:

Materials

SDS-PAGE:

• NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.5 mm, 10-well (Separation Range: 3.5 to 260 kDa) (Thermofisher Scientific #NP0335BOX)

• NuPAGE™ MOPS SDS Running Buffer (20X) (Thermofisher Scientific # NP0001)

• SeeBlue™ Plus2 Pre-stained Protein Standard (Thermofisher Scientific # LC5925)

• NuPAGE™ LDS Sample Buffer (4X) (Thermofisher Scientific #NP0007)

• NuPAGE™ Sample Reducing Agent (10X) (Thermofisher Scientific #NP0004)

• XCell SureLock™ Mini-Cell (Thermofisher Scientific #EI0001)

Primary Antibodies:

• CDK5 (D1F7M) Rabbit mAb (Cell Signaling Technology #14145)

• p35/25 (C64B10) Rabbit mAb (Cell Signaling Technology #2680)

• p39 Antibody (Cell Signaling Technology #3275)

Detection:

• Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) (Millipore-Sigma #GENA934)

• Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep) (Millipore-Sigma # GENA931)

• ECL^™ Western Blotting Reagents (Millipore-Sigma #GERPN2106) Follow manufacturer’s instructions

Wash Buffer: 1X TBS, 0.1% Tween^® 20 (Millipore Sigma #P9416),

Blocking Buffer: 5% BSA (Millipore Sigma # A4503) or Powdered Milk (LabScientic # M0842) prepared in the wash buffer.

Equipment:

• Thermomixer™ R (Eppendorf™ #05–400-205) or equivalent

• Refrigerated Micro Centrifuge (Eppendorf™ #5417R) or equivalent

• PowerPac™ Basic Power Supply (Bio-Rad #1645050) or equivalent

• Imager (Fluorochem M System Bio-Techne #92–15312-00) or equivalent

• Alternatively for film:

• HyBlot CL® Autoradiography Film (Denville Scientific #E3018) or equivalent

• Film Cassette (Amersham Hypercassette Autoradiography Cassettes #RPN11649)

• (requires a darkroom and developer [Medical Film Processor # SRX-101A])

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• MμltiFlex™ Pipet Tips, Sorenson BioScience (VWR #53550–167)

• For gel loading

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Western Blot Boxes (Alkali Scientific #BC1200–5 or BC1200–7) or equivalent

• Ice Bucket

• Gel Knife (Thermofisher Scientific #EI9010)

• Heavy-Duty Long-Blade Scissors (Fisher Scientific #14–275C)

• Filter forceps, blunt end (Millipore Sigma #XX6200006P)

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

Thaw selected protein lysates (stored at −80°C) but keep on ice once melted. Denature samples using the composition below:

Sample	x μl
NuPAGE™ LDS Sample Buffer (4X)	2.5 μl
NuPAGE™ Sample Reducing Agent (10X)	1.0 μl
Deionized Water	to 6.5 μl
Total Volume

For dilute protein samples, the above volumes can be scaled up as appropriately needed, but the maximum load volume per well is ~ 37 μl.

Heat samples at 70˚C for 10 minutes using a thermomixer, then place on ice. Give protein samples a quick spin in a microcentrifuge before loading for electrophoresis.

Prepare 1X MOPS SDS Running Buffer (~1L). Prepare gel by removing the comb, rinsing the gel wells, removing the white tape at the bottom of the gel cassette, and placing gel into mini gel tank. Fill the lower and upper chambers of the XCell SureLock™ Mini-Cell apparatus as recommended.

Load samples. Ensure to leave one well for the protein marker. Gels can be run at 200 V constant for 50 minutes, although lower voltages (with longer run times) can be used.

Gel electrophoresis and protein separation are then followed by membrane transfer and antibody detection. The details of immunoblotting are described in UNIT 6.2 (Ni, D., Xu, P., and Gallagher, S., 2017).

Dilute primary antibodies 1:500 – 1:1000 to detect Cdk5, p35, and p39 (Cell Signaling recommends diluting the primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween^® 20). Incubate the membrane overnight at 4°C.

A proper protein control (i.e., GAPDH, Beta-Actin, or α-Tubulin) should also be measured to ensure uniform loading. If using chemiluminescent-based antibody detection, the membrane will need to be stripped following the detection of Cdk5 or its activators. To strip the membrane, we use either Re-Blot Strong Antibody Stripping Solution 10x (Millipore-Sigma #2504) or Restore™ PLUS Western Blot Stripping Buffer (ThermoFisher Scientific #46430). Follow the manufacturer’s instructions. The membrane can then be reprobed for the preferred protein loading control - both Beta-Actin Proteintech, Inc. #66009) or Anti-α-Tubulin Millipore-Sigma #T9026) antibodies work well (Figure 7).

Figure 7.

Western blot for Cdk5 and its activator p35 along with Beta-actin as a protein loading control. The brain of the Tgp35 shows higher expression levels of p35 due to the transgene as compared to the WT littermate control, while Cdk5 expression levels were unaffected. The p35 null mice (p35−/−) show targeted genetic depletion of p35 in contrast to the WT mouse.

SUPPORT PROTOCOL 3:

IMMUNODETECTION ANALYSIS FOR CDK5, P35, AND P39

Along with Western blotting, the immunodetection of Cdk5, p35 and p39 by immunofluorescence (IF) can be useful as a means to analyze protein abundance, distribution, and cellular localization (Contreras-Vallejos et al., 2014; Utreras et al., 2009a; Utreras et al., 2009b; Utreras et al., 2014; Rozas et al., 2016; Coddou et al., 2017; Sandoval et al., 2018). Detailed immunostaining protocols are available at www.cellsignal.com, www.abcam.com, and vectorlabs.com. Here, we provide techniques specific for immunodetection of Cdk5, p35 and p39 in either cultured cells (Figure 8 and 9) or neuronal tissues (Figure 10 and 11).

Figure 8.

Representative IF for p35 (red) in transfected COS7 cells by using three diferent antibodies against p35: SantaCruz Biotechnology anti-p35 antibodies C19 and N20 and anti-p35 C64B10 from Cell Signaling Technology. DAPI was used as nuclear stain (white). We observed cytoplasmic distribution of p35 in the transfected COS7 cells. Yellow bar = 20 μm.

Figure 9.

Representative ICC images for p35 (red) in COS7 cells transfected with a p35 expression vector (left panels), B104 cells (middle panels), and cultured trigeminal neurons (right panels). Microtubules were stained with either α-tubulin (COS7 and B104 cells) or βIII-tubulin (neurons) (green). Yellow bar = 20 μm.

Figure 10.

Representative IF against Cdk5 (left panel), p35 (middle panel) and p39 (right panel) from an adult mouse trigeminal ganglion. βIII-tubulin (red) was used as a neuronal marker (red) and DAPI a nuclear stain (white). We detected cytoplasmic distribution of endogenous levels of p35, p39 and Cdk5 in mouse trigeminal ganglion by IF. Yellow bar = 50 μm.

Figure 11.

Representative IF against P2X2 (red), Cdk5 (green), βIII-tubulin (white), and DAPI (cyan) from a section of a rat nodose ganglia. Previously, we reported that P2X2aR colocalized with both Cdk5 and p35 close to the plasma membrane in transfected HEK293 cells and showed that Cdk5 can then phosphorylate P2X2R (Coddou et al, 2017). In this section of the rat nodose ganglia, we see that Cdk5 co-localizes with P2X2R in the ganglionic neurons. Yellow bar = 50 μm.

Immunofluorescence detection of p35:

Materials

Cell line of interest

Precision cover glasses (Deckglaser cover glasses, Superior Marienfeld)

Fixative: 4% paraformaldehyde and 4% sucrose solution

• PFA: Electron Microscopy Sciences 16% Paraformaldehyde Aqueous Solution (Fisher Scientific # 50–980-487)

• Sucrose, molecular biology reagent, >99% (MP Biomedicals)

Permeabilization Solution:

• Methanol (HPLC), (Fisher Scientific #A452–4)

• 0.1% PBS–Triton X-100 (Millipore Sigma #X100)

Blocking Buffer: 5% BSA-PBS (BSA: Millipore Sigma # A4503)

Primary Antibodies (Diluted in 1% BSA-PBS):

• p35/25 (C64B10) Rabbit mAb (Cell Signaling Technology #2680). Note: Santa Cruz Biotechnology anti-p35 antibodies (C19) (#sc-820) and (N20) (#sc-821) previously used for immunostaining have been discontinued.

• Anti-βIII Tubulin mAb (Promega #G7121)

• Anti-α-Tubulin antibody, Mouse monoclonal (Millipore Sigma #T6199)

Secondary Alexa Fluor antibodies are available from Thermofisher Scientific. Select a fluorophore that both matches your planned experimental designs and is compatible with your microscope setup.

DAPI Solution (Thermofisher Scientific #62248)

Mounting medium: FluorSave™ Reagent (Millipore Sigma # 345789)

Confocal Microscope (LSM 710 Meta Model; Carl Zeiss Microscopy) or equivalent

Images were processed using the LSM Image Browser software (Carl Zeiss Microscopy) or similar software.

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Ice Bucket

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

The expression of p35 was examined in transfected COS7 (African green monkey kidney) cells, B104 (Rat neuroblastoma) cells, and in cultured mouse primary trigeminal ganglia (TG) neurons (Figure 8 and 9). Three different anti-p35 antibodies were tested: C19 and N20 from Santa Cruz Biotechnology (no longer commercially available) and anti-p35 C64B10 from Cell Signaling Technology. COS7 cells normally do not express endogenous p35, so cells were transfected with a CMV-p35 plasmid for 24 hours. The rat B104 neuroblastoma cell line and cultured mouse TG neurons endogenously express p35. Along with p35, cells were also stained for either α-tubulin (cultured cell line) or βIII-tubulin (trigeminal neurons).

1. Seed desired cell line or primary mouse TG neurons onto a precision cover glass in a 24-well culture plate.

2. Once at the desired confluency, discard the culture medium. Then wash the cells with 1X PBS for 1 – 2 min at room temperature.

3. Fix cells with 400 μl of 4% PFA/4% sucrose solution for 20 min at room temperature.

4. Wash 3 times with 1X PBS (5 min each) at room temperature

5. Permeabilize with 500 μl of 0.2% TritonX-100 solution for 5 min at room temperature

6. Wash 3 times with 1X PBS (5 min each) at room temperature

7. Add blocking solution (BSA 5% in 1X PBS) for 1 h at room temperature

8. Prepare the primary Ab in 1% BSA - 1X PBS solution.

9. Add necessary volume (~80 – 120 μl) of primary antibody onto cover glass. Incubate overnight at 4°C.

10. Wash 3 times with 1X PBS at room temperature for 5 min

11. Prepare the secondary Ab (1: 500) plus DAPI (1:1000) in 1% BSA - 1X PBS solution.

12. Add necessary volume (~80 – 120 μl) of secondary antibody over cover glass. Place into a humid chamber and incubate for 2 hours at room temperature.

13. Wash 3 times with 1X PBS (5 min each) at room temperature

14. Wash covers for 5 min with dH₂O.

15. Mount with FluorSave, avoid bubbles.

16. Let dry slides at room temperature for couple of hours and store at 4°C.

Immunofluorescence (IF) detection of Cdk5, p35 and p39 in tissues:

Materials

Tissue of Interest

For mouse neuronal tissue, whole animal perfusion with a 4% paraformaldehyde (PFA) solution is recommended to provide uniform fixation (Gage et al., 2012).

PFA - Electron Microscopy Sciences 16% Paraformaldehyde Aqueous Solution (Fisher Scientific # 50–980-487)

Sucrose, molecular biology reagent, >99% (MP Biomedicals)

Tissue-Tek® O.C.T. Compound, Sakura® Finetek (Optimal Cutting Temperature Compound) (VWR #25608–930)

Tissue-Tek® Cryomold® Molds Sakura® Finetek (VWR #25608–916) or equivalent

Fisherbrand™ Superfrost™ Plus Microscope Slides (Fisher Scientific 12–550-15)

Leica CM3050 S Cryostat

Microscope slide box

Super PAP Pen (Thermofisher Scientific #008899)

StainTray™ Slide Staining System (Thomas Scientific # 1219D69) or equivalent

Permeabilization / blocking buffer: 5% BSA + 0.3% PBS–Triton X-100

• Triton X-100 (Millipore Sigma #X100)

• BSA (Millipore Sigma # A4503)

Primary Antibodies:

• Cyclin-dependent kinase 5 (Cdk5) Antibody (Phosphosolutions #308-CDK5) or Anti-Cdk5 Antibody (DC 17) (Santa Cruz Biotechnology # sc-249) (1:100 Dilution in blocking buffer)

• p35/25 (C64B10) Rabbit mAb (Cell Signaling Technology #2680) (1:100 Dilution)

• p39 Antibody (Cell Signaling Technology #3275) (1:100 Dilution)

• Anti-βIII Tubulin mAb (Promega #G7121) (1:1000 Dilution)

Secondary Alexa Fluor antibodies are available from Thermofisher Scientific. Select a fluorophore that both matches your planned experimental designs and is compatible with your microscope setup.

DAPI Solution (Thermofisher Scientific #62248)

FluorSave™ Reagent (Millipore Sigma # 345789)

Confocal Microscope (LSM 710 Meta Model; Carl Zeiss Microscopy) or equivalent

Images were processed using the LSM Image Browser software (Carl Zeiss Microscopy).

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Ice Bucket

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

Fixation and Cryosectioning

• 1Remove the tissue of interest and immerse in fixative (4% PFA in 1X PBS). Add about 10X the volume of fixative per volume of tissue for adequate fixation. Leave the tissue to fix for 2 h at room temperature.
• 2Discard the 4% PFA in a special PFA waste container, then add an equivalent volume of 30% sucrose solution (cryoprotective solution). Incubate the tissue in 30% sucrose at 4°C for 24 – 72 h or until the tissue settles to the bottom of the tube.
• 3Discard the 30% sucrose and blot the tissue gently to remove excess solution from the tissue.
• 4Add a little O.C.T. to a cryomold and leave at −20 to −80°C for a few minutes, until a frozen layer forms.
• 5Deposit the tissue on top of the frozen O.C.T. layer and immediately add more O.C.T. until it is completely covered (avoid generating bubbles). The correct orientation of the tissue in the cryomold is typically needed for proper and consistent tissue sections, particularly with brain and trigeminal ganglion.
• 6Freeze the tissue in OCT at −80°C for 1 – 2 h.
• 7Remove the embedded tissue from the cryomold and cut on a cryostat. It is recommended to cut 12 – 16 μm sections. Turn on the cryostat 3–4 hours before use, so that it reaches a temperature of −20°C.
• 8Store sections of tissues in a slide box at 4°C or use for IF immediately.

For cyrosectioning see https://www.leicabiosystems.com/knowledge-pathway/the-art-of-embedding-tissue-for-frozen-section/ by Dr. Stephen Peters.

Immunofluorescence in tissue sections

• 9Remove the excess O.C.T. that is around each tissue section with the help of a fine-tip forceps, taking care not to tear or remove the tissue adhered to the slide. An alternative way to remove the O.C.T. is to immerse the slide in distilled water (in a 50 ml falcon tube) for 1 min, then immerse in 1X PBS for 1 – 2 min at room temperature.
• 10Draw a circle around the tissue section on the slide with a PAP pen.
• 11Permeabilize and block the tissue with a 5% BSA + 0.3% Triton X-100 solution in a slide tray. Incubate at room temperature for 1 ½ hr. A volume of 80 – 120 μl is typically needed to cover the tissue, depending on its size.
• 12Discard the blocking and permeabilization solution.
• 13Wash the tissue 2X - Cover the tissue with 1X PBS (~ 1000 μl) for 5 min at room temperature. Avoid touching the tissue with the pipette.
• 14Prepare the primary Ab solution in 5% BSA + 0.3% Triton X-100 solution.
• 15Add the necessary volume (~80 – 120 μl) to completely cover the tissue and incubate the slide sections at 4°C overnight in a slide tray. Be careful when moving the slide tray to 4°C to ensure that the primary Ab solution does not flow off the tissue during transportation.
• 16Discard the primary Ab solution and perform 3 washes for 5 min with 1X PBS.
• 17Prepare the secondary Ab solution (for AlexaFluor Ab, use ~1:500 dilution) in 5% BSA + 0.3% Triton. DAPI can be added to the secondary Ab solution (~1:1,000 dilution).
• 18Incubate the tissue sections with the secondary Ab solution in the slide tray for 2 h at room temperature (hereafter, avoid exposing the samples to light to prevent photobleaching).
• 19Carry out 3 washes with 1X PBS for 5 min each in the slide tray. Run a final wash with distilled water for 5 min.
• 20Clean the coverslips with 70% EtOH and allow to dry. Mount tissue sections with 20–30 μl of FluorSave.
• 21Place the slides in the dark and allow the FluorSave to dry (~1–3 hours) at room temperature. Slides can be kept in a slide box at 4°C for several days (Figure 10 and 11).

SUPPORT PROTOCOL 4:

GENETICALLY ENGINEERED MICE (+ AND - CONTROLS)

Numerous genetically engineered mouse models have been generated to better understand the molecular role of Cdk5 in neuronal activity. Ablation of Cdk5 leads to embryonic lethality (Ohshima et al., 1996), while p35−/− mice are viable but still display cortical layering defects (Chae et al., 1997, Ohshima et al., 2001). The p39−/− mice, however, show no observable phenotypic neurological defects and no significant decrease in Cdk5 activity in neuronal tissues such as the brain and trigeminal ganglia (Ko et al., 2001; Prochazkova et al., 2017). Compound deletion of both p35 and p39 is needed to fully replicate the phenotype of the Cdk5 knockout mice. Because of the embryonic lethality associated with Cdk5 deletion, Cdk5 flox mice were therefore developed (Hirasawa et al., 2004). Transgenic overexpression of the Cdk5 activators such as p35 and the truncated form p25 alternatively provide a means of genetically triggering increased Cdk5 activity in a mouse. Primarily, behavioral abnormalities have been detected in transgenic overexpressing p35 mice (Takahashi et al., 2005) but overexpression of p25, in contrast, leads to neurodegeneration that mimics Alzheimer’s disease (Cruz et al., 2003). Transgenic overexpressing p35 mice (Tgp35) have a 50% neuronal increase in Cdk5 activity (Takahashi et al., 2005, Prochazkova et al., 2013) without the pathology seen by overexpressing p25, so these Tgp35 mice can be useful as a positive control when running the above kinase activity assay. Conversely, p35−/− mice neuronally display significantly reduced Cdk5 activity (Chae et al., 1997, Ohshima et al., 2001), yet are still viable and can, therefore, be readily used as a negative control for measuring Cdk5 activity in mice.

Genotyping Transgenic p35 mice

Transgenic p35 overexpressing mice (Tgp35) can be used as a positive control for measuring Cdk5 activity, particularly when investigating the Cdk5 activity in other experimental mouse models. Tgp35 should show increased Cdk5 activity compared to a littermate wild-type control and can serve as a means to check proper immunoprecipitation of Cdk5. To generate the Tgp35 mouse, a cloned 1.2 kb mouse p35 promoter fragment was used to drive the expression of the mouse p35 (Cdk5r1) gene (Takahashi et al., 2005). An SV40 tag in the transgene also contains a SpeI (a/ctagt) restriction enzyme site for genotyping (Figure 12). Tgp35 mice can be useful to study the effects of Cdk5 hyperactivity on mouse behavior.

Figure 12.

PCR results for Tgp35 and WT mice. After the SpeI digest, mice hemizygous for the p35 transgene show 3 bands on a gel: a 760 bp band from the wild type allele and two SpeI cut bands 483 and 276 bp derived from PCR amplification of the transgenic allele. Note: Generation of transgenic mice via microinjection results in the random integration of the transgene into the mouse genome (Cho et al., 2009). Typically, we do not breed mice to homozygosity in case the integration of the transgene occurred within an unknown genomic element of possible consequence. Besides Tgp35 mice, cultured cells transfected with a p35 expression vector may also be used as a positive control (see Critical Parameters below).

Materials

For genotyping purposes, we generally use TaKaRa Ex Taq® DNA Polymerase (# RR001B), but any preferred PCR Taq DNA polymerase will probably work.

PowerPac™ Basic Power Supply (Bio-Rad #1645050) or equivalent

GeneAmp™ PCR System 9700 (Thermofisher Scientific #4339386)

Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

Pipetman P1000 or equivalent

Pipetman P200 or equivalent

Pipetman P100 or equivalent

Pipetman P20 or equivalent

Pipetman P10 or equivalent

Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

ProCycle™ 0.2mL StableStrip™ 8-Strip PCR Tubes (Alkali Scientific #PC7061-A) or equivalent

Water, PCR Qualified (Quality Biological # 351–161-671) or equivalent

UltraPure™ Agarose (Thermofisher Scientific #16500500) or equivalent

Primers can be synthesized by IDT technologies or other oligo providers.

Forward Primer 12F	: 5’ ACA TCC TGC CAC GGT GAC 3’
Reverse Primer 10R	: 5’ AGT ATC GGA TGT ACA GCT ATG 3’

SpeI (Millipore-Sigma #11008951001)

PCR Reaction

Distilled Water	34.75	μl
10 × Buffer	5.0	μl
dNTP Mixer 2.5 mM	4.0	μl
Forward Primer 12F (10 μM)	1.0	μl
Reverse Primer 10R (10 μM)	1.0	μl
TaKaRa Ex Taq®	0.25	μl

Reaction mix: 46 μl

Template: 4 μl

Cycling Conditions

Step 1

97°C: 3 min

Step 2

94°C: 1 min

60°C: 30 sec

72°C: 1 min

Set up for 35 cycles

Step 3

72°C: 10 min

Step 4

4°C: ∞

Amplicon Size: 760 bp

When the PCR is finished, run 30 μl of the PCR product on a 2% agarose gel to check PCR amplification. Digest the remaining PCR product with SpeI to identify the transgenic mice:

Buffer H	2.5	μl
SpeI	1.0	μl
Water	1.5	μl
PCR product	20.0	μl

Incubate samples at 37°C for 1–4 h and then run the PCR product on a 2% agarose gel with a 100 bp ladder. If the sample is the p35 transgenic, you will get 3 bands: 760, 483 and 276 bp. If the sample is the wild-type mouse, you only get 1 band of 760 bp (Figure 12).

p35 Knockout

Two p35 null mice were generated (Chae et al., 1997, Ohshima et al., 2001) using conventional gene targeting techniques via homologous recombination (Hall et al., 2009). The mice, therefore, have a knocked-in neomycin resistance cassette to disrupt the p35 gene (sequence matching the neomycin resistance gene was used to create a primer needed to detect the mutant allele) (Figure 13). The p35 knockout mice can be useful as a negative control for the Cdk5 assay with an 80–90% decrease in kinase activity and can also be used to study the effects of Cdk5 hypoactivity on mouse behavior (opposite of the Tgp35 mice) (Prochazkova et al., 2013). The genotyping protocol below follows the p35 null mice generated by Dr. Li-Huei Tsai’s lab. These mice are available through Jackson Labs (B6.129S4-Cdk5r1^tm1Lht/J: Stock No: 004163 | p35-).

Figure 13.

PCR was used to detect p35−/− mice. One of the primers binds to the Neo^r cassette to generate a 1,300 bp PCR product from the targeted allele, while the wild type band is 750 bp. The p35+/− mice are bred together to produce the p35−/− mice (~25%) along with heterozygous p35+/− mice (~50%) and homozygous wild type mice (~25%).

p35 Up	: 5’ ACG CAG ATC CGC AGG ACT AAA C 3’
p35 Down	: 5’ CAG AGC ATG TAG AGG AAG ACC ACA 3’
p35 Neo	: 5’ GGA GAG GCT ATT CGG CTA TGA C 3’

PCR Reaction

Distilled Water	32.75	μl
10 × Buffer	5.0	μl
dNTP Mixer 2.5 mM	4.0	μl
p35 Up (10 μM)	2.0	μl
p35 Down (10 μM)	1.0	μl
p35 Neo (10 μM)	1.0	μl
TaKaRa Ex Taq®	0.25	μl

Reaction mix: 46 μl

Template: 4 μl

Cycling Conditions

Step 1

97°C: 3 min

Step 2

94°C: 1 min

62°C: 1 min

72°C: 2 min

Set up for 35 cycles

Step 3

72°C: 10 min

Step 4

4°C: ∞

Run PCR product on 1.2% agarose gel with a 100 bp ladder (Figure 13).

Results:

+/+	750 bp band
−/−	1,300 bp band
+/−	both bands

Cdk5 Knockout

The Cdk5 null mice display perinatal lethality, so, to use these mice for Cdk5 studies, pregnancies must be timed, and embryos are generally collected at E18.5. The embryonic Cdk5−/− brain can be studied to determine the effects of Cdk5 deletion (Contreras-Vallejos et al., 2014) and can be used as a negative control in the Cdk5 assay. Cdk5 null brain tissue can also be used as an immunoprecipitation negative control as seen in Pareek et al., (2007) (Figure 14).

Figure 14.

Cdk5 wildtype and knockout mice can be used to confirm Cdk5- mediated phosphorylation of a potential substrate. The thermosensitive transient receptor potential channel TRPV1 was identified as a Cdk5 substrate and was immunoprecipitated from NIH3T3 +TRPV1 cells and DRG cultures. The immunoprecipitated TRPV1 was then used as a substrate in the Cdk5 kinase assay. Cdk5 immunoprecipitated from the wild-type mice was able to phosphorylate TRPV1, while only minimal background radioactivity is seen with the brain lysates from the Cdk5 null mouse (Pareek et al., 2007). Copyright (2007) National Academy of Sciences, U.S.A.

Cdk5 For	: 5’ ATT GTG GCT CTG AAG CGT GTC 3’
Cdk5 Rev	: 5’ CTT GTC ACT ATG CAG GAC ATC 3’
PGK-1	: 5’ CCA TCT GCA CGA GAC TAG T 3’

PCR Reaction

Distilled Water	28.8	μl
10 × Buffer	5.0	μl
dNTP Mixer 2.5 mM	4.0	μl
Cdk5 For (10 μM)	4.0	μl
Cdk5 Rev (10 μM)	2.0	μl
PGK-1 (10 μM)	2.0	μl
TaKaRa Ex Taq®	0.2	μl

Reaction mix: 46 μl

Template: 4 μl

Cycling Condition

Step 1

97°C: 3 min

Step 2

94°C: 30 sec

55°C: 1 min

77°C: 1 min

Set up for 30 cycles

Step 3

72°C: 10 min

Step 4

4°C: ∞

Run PCR product on 2% agarose gel with 100 bp ladder (Figure 15).

Figure 15.

PCR to identify the targeted Cdk5 null allele. Ablation of Cdk5 results in perinatal lethality so embryos were collected at E18.5. PCR generates a 500 bp PCR product for the null allele and a 309 bp wild-type band.

Results:

−/−	500 bp band
+/+	309 bp band
+/−	both bands

BASIC PROTOCOL 2:

IDENTIFYING NEW CDK5 SUBSTRATES AND KINASE INHIBITORS

Reversible phosphorylation has a central part in regulating cell activity by modulating a protein’s activity, stability, and ability to bind with other proteins (Cohen, 2002). Abnormal phosphorylation has also been implicated in various human diseases. Cdk5 is a multifunctional protein kinase with key roles in neurodevelopment as well as neurodegenerative diseases, while recently also being recognized for its involvement in non-neuronal cellular activities, such as cancer progression and immune responses (Contreras-Vallejos et al., 2012). To better determine Cdk5’s mechanistic role in a particular cellular process, key downstream phosphorylated substrates need to be identified. Below is a protocol that provides a method to identify Cdk5 substrates.

As described above, numerous Cdk5 substrates have been identified since its discovery, including DARPP32, Tau, MAP2, NFH/M, Nudel, STAT3, β-catenin, amphyphysin, dynamin I, synapsin 1, FAK, Munc-18, and the NMDA receptors NR2A and NR2B. A few known Cdk5 substrates can also be phosphorylated by Cdk1 or Cdk2, including Histone H1, which has traditionally been used as a readily available substrate for measuring Cdk5 activity (see below) (Dhavan and Tsai, 2001). Cdk5 is a proline-directed Ser/Thr kinase, which, at its simplest, comprises an (S/T)P phosphorylation site, but 49% of Cdk5 substrates typically have a canonical (S/T)PX(K/H/R) phosphorylation motif that includes a basic amino acid residue in position +3 (Borquez et al., 2013). Identification of new potential Cdk5 substrates can be conducted using Scansite (https://scansite4.mit.edu/4.0/#home), which has a built-in search sequence for Cdk5 phosphorylation sites (Obenauer et al., 2003). Increased use of mass spectrometry to identify phospho-proteins in conjunction with immobilized metal affinity chromatography has also allowed for the detection of new potential Cdk5 substrates (Contreras-Vallejos et al., 2014). Candidate Cdk5 substrates can be tested using either recombinant proteins or with peptides containing the suspected Cdk5 phosphorylation site. The following protocol is geared towards using peptides to identify potential Cdk5 substrates:

Materials

Cdk5 Substrate:

• 10 amino acid peptides containing a suspected (S/T)P Cdk5 Site (Use a preferred peptide synthesis service: i.e., Peptide 2.0, Chantilly, VA, 21^st Century Biochemicals, Marlboro, MA, GenScript, Piscataway, NJ). Peptides should be made to >95% purity. Peptides are diluted in ultrapure water to a 10mM concentration for use in the Cdk5 assay.

• Histone H1 (1 mg/ml) (Sigma #H5505–25mg)

• NF-H peptide (VKSPAKEKAKSPVK)– Alternative control Cdk5 substrate rather than Histone H1

• CDK5/p35: active, GST tagged human (Sigma #SRP5011–10μg)

• Or

• CDK5/p25: active, GST tagged human (Sigma # C0745–10μg)

• 75 mM phosphoric acid (Sigma-Aldrich # 345245) (Use 5.1 ml of Orthophosphoric acid diluted into water to make 1L of 75mM phosphoric acid)

• 95% ethanol (Warner and Graham # 200196)

• Grade P81 Ion Exchange Paper, sheet, 460 × 570 mm

• P81 phosphocellulose from GE Healthcare (3698–915) has been discontinued.

• P81 phosphocellulose is available from:

• Prof Jonathan Oakhill, St. Vincent’s Institute of Medical Research

• 9 Princes St, Fitzroy VIC 306, Australia (New source)

• joakhill@svi.edu.au

• P81 produced in the lab is indistinguishable from the GE Healthcare product

• LSA-50 paper has also been suggested as an alternative to P81 phosphocellulose paper (Appelmans et al. 2021).

Radioactivity (working with ATP [γ−³²P]):

• follow your institute’s guidelines for the proper use and handling of radioactive material

• Nalgene™ Acrylic Benchtop Beta Radiation Shield (Thermofisher Scientific # 6700–1812 or 6700–2418) or equivalent

• 5 Gallon Carboy with Handles (Research Product International #2210–0050) or equivalent

• Funnel

• Rad-3 Lock Box, 5 Gallon Carboy Capacity (Research Product International #RLB-03) or equivalent

• Model 3 General Purpose Survey Meter (Ludlum Measurements, Inc #48–1605) or equivalent

• Model 44–9 Alpha-Beta-Gamma Detector (Ludlum Measurements, Inc #47–1539) or equivalent

• Defenders® 5 Gallon Step Can (Rubbermaid Commercial Products # FGST5EGLRD) or equivalent

• Rad-4 Lock Box, Large Capacity Unit (Research Product International #RLB-04) or equivalent

• Corrugated Waste Box for Radioactive Material (Research Product International #BRS-15) or equivalent

• Heavy Duty Poly Liners (Research Product International #BRS-16) or equivalent

• Caution Radioactive Material Tape (Research Product International #140046) or equivalent

• COUNT-OFF Surface Cleaner (PerkinElmer #6NE942T) or equivalent

Bio-Safe II Complete Counting Cocktail (Research Product International # 111195)

KIMBLE® 20 mL Glass Scintillation Vial (DWK Life Sciences Duran Wheaton Kimble # 74500–20) or equivalent

Scintillation counter (Hidex 300 SL Standard #425–201) or equivalent

Lab Equipment:

• Thermomixer™ R (Eppendorf™ #05–400-205) or equivalent

• Norlake Freezer −20 (Thomas Scientific #1156Y04) or equivalent

• HB-500 Minidizer Hybridization Oven (Lab Rep Co #95–0330-01)

• Centrifuge (Eppendorf™ #5415C) or equivalent

• Refrigerated Micro Centrifuge (Eppendorf™ #5417R) or equivalent

• IBI Scientific™The Belly Dancer™Orbital Platform Shaker (Fisher Scientific

• #15–453-211) or equivalent

• Mini Vortex-Genie 2 (Daigger #G22220) or equivalent

• Sample mixer for rotation of tubes (e.g., HulaMixer™ Sample Mixer Thermo Scientific #15920D)

Lab Supplies:

• Nitrile gloves (Halyard Purple Nitrile gloves powder free) or equivalent

• Pipetman P1000 or equivalent

• Pipetman P200 or equivalent

• Pipetman P100 or equivalent

• Pipetman P20 or equivalent

• Pipetman P10 or equivalent

• Filter pipette tips (Denville Low Retention Pipet Tips, Thomas Scientific) or equivalent

• Portable Pipet-Aid (Drummond #4–000-100) or equivalent

• Serological Pipets (Corning Costar Stripette™ - 5ml #4051, 10ml #4101, 25ml #4251) or equivalent

• 1.7 mL Microcentrifuge Tubes (Crystalgen #L-2052) or equivalent

• Nalgene™ Microcentrifuge Tube Rack (Thermofisher Scientific #5973–0015) or equivalent

• 80-Place Tube Rack (Thomas Scientific #1159V62) or equivalent

• Multi Tube Rack (Boekel Scientific #120008) or equivalent

• Falcon™ 15 mL Centrifuge Tubes (Fisher Scientific #14–959-49B) or equivalent

• Falcon™ 50 mL Centrifuge Tubes (Fisher Scientific #14–432-22) or equivalent

• Heavy-Duty Long-Blade Scissors (Fisher Scientific #14–275C)

• Lab Soaker Bench Protector Mat (Daigger Scientific #NAL74018) or equivalent

• Kimwipes (Kimberly Clark Professional Kimtech #5511) or equivalent

• Paper Towels

• Deionized H₂O

Protocol Steps

1. Make a 1:25 dilution of CDK5/p35 in 1X Kinase Buffer

2. Add the following mixture into a labeled microfuge tube:

   • 10 μl KB5X
   • 10 μl CDK5/p35 (1:50)
   • 5 μl Peptides (10mM)
   • q.s. with water to a 40 μl volume
   • Be sure to include a Negative Control (without CDK5/p35 and/or a substrate) and a Positive Control (with Histone H1 – 1 mg/ml)

Sample Experiment with test peptides:

Reagent	Peptides	Positive Control	Neg Con (Enz)	Neg Con (Substrate)
5X Kinase	10 μl	10 μl	10 μl	10 μl
Histone H1 (10 μg)	-	10 μl	10 μl	-
Enzyme 1:50	10 μl	10 μl	-	10 μl
Peptide	5 μl	-	-	-
Water	15 μl	10 μl	20 μl	20 μl
ATP Mix* (added last)	10 μl	10 μl	10 μl	10 μl

Can make a master mix of KB5X and CDK5/p35- Add 20 μl to the appropriate tubes. For the Positive Control, add 10μl of histone solution (1 mg/ml)

Work with ATP [γ−³²P] should be performed in a designated radioactive room. When detecting Cdk5 activity using a scintillation counter, samples are generally run-in triplicate.

• 6Mix all the reagents in the tubes by vortexing, then add 10 μl of the hot ATP mix (see Reagents and Solutions), again mix thoroughly and give a quick spin to collect the contents to the bottom of the tube.
• 7Incubate at 30°C for 60 min in an incubator.
• 8Stop the reaction by spotting 20 μl aliquots of the reaction mixture onto P81 phosphocellulose squares. Can mark out and label about 1-inch squares. Let it air dry.
• 9Wash 3X for 15 min each in 75 mM phosphoric acid. Place P81 phosphocellulose strips in a 2L plastic beaker and cover phosphoric acid solution. Place on rotator during washes.
• 10Wash once in 95% ethanol, then air dry.
• 11Cut squares and transfer into vials containing 15 ml Bio-Safe II scintillation fluid for counting on a Hidex 300 SL scintillation counter (Figure 16). Alternatively, one may be able to get away with water rather than scintillation fluid, although the cpm is reduced. Can label scintillation vials.

Figure 16:

A peptide kinase assay can be used to pinpoint the most probable Cdk5 phosphorylation sites on a protein. Six putative Cdk5 sites were identified on the cytoplasmic N-terminal domain of the chemosensor TRPA1. Ten amino acid peptides were incubated with recombinant Cdk5 and ATP [γ−³²P]. Peptides for Ser⁴⁴⁹ and Thr⁴⁸⁵ were most highly phosphorylated. (Hall et al. 2018).

Note: The above protocol is tailored for use with small peptides that are not amenable for SDS-PAGE, but a recombinant full-length protein (~.5 to 1.5 μg), an immunoprecipitated protein (Pareek et al., 2007; Hall et al., 2018), or a large peptide domain can alternatively be tested as a substrate for Cdk5, particularly to determine if the candidate Cdk5 site within the protein substrate is accessible for phosphorylation. After incubating the test substrate with recombinant Cdk5 and ATP (γ−³²P) for 60 min at 30°C, 20 μl of the kinase reaction can be spotted onto P81 phosphocellulose squares (Steps 5–8; Basic Protocol 2). Alternatively, the test immunoprecipitated or recombinant protein can be electrophoresed on a gel and radioactivity detected using autoradiography film (Steps 21–31; Basic Protocol 1) (peptides > 2kDa can be electrophoresed if using the appropriate gel).

Identifying new kinase inhibitors

Note: The peptide kinase assay can be readily adapted to test for new kinase inhibitors using the recombinant Cdk5.

As mentioned, abnormal phosphorylation can have a role in various human diseases. Cdk5 hyperactivity has been associated with neurodegenerative diseases, chronic pain, and cancer progression. The development of safe and effective Cdk5 inhibitors has therefore been a current therapeutic goal to treat these conditions. The following protocol can be used to test for new Cdk5 inhibitors.

Essentially, three main strategies have been employed to inhibit pathological Cdk5 activity (Gomez et al., 2020):

1. Small molecules that compete for the active ATP binding site within the kinase domain, including roscovitine and olomoucine. These Cdk5 inhibitors, however, can often non-specifically affect other Cdks essentially because of their shared sequence homology.

2. Peptides derived from truncated portions of p25 that interfere with the formation of Cdk5/p25 complexes. These peptides include the Cdk5 inhibitory peptide (CIP) (amino acids 154–279) (Amin et al., 2002; Zheng et al., 2002) and p5, a 24-residue peptide that spans CIP residues 245–277 (Figure 17 and 18) (Zheng et al., 2010).

3. Small molecule inhibitors like tamoxifen that disrupt Cdk5/p25 binding (Corbel et al., 2015). A bimolecular fluorescence complementation screening assay was also recently developed to identify small molecules that block Cdk5/p25 protein-protein interactions (Bellón-Echeverría et al., 2018).

Figure 17.

p5, a 24 a.a. peptide derived from p25 was identified as an inhibitor of Cdk5 activity. p25 truncated peptides were tested for their inhibitory properties. p5 was the shortest and best inhibitor of Cdk5-p25 in an in vitro assay with Histone H1 as substrate (Zheng et al., 2010).

Figure 18.

p5 equally inhibits both Cdk5-p25 and Cdk5-p35 activities. Increasing concentrations of p5 were incubated with either Cdk5/p35 or Cdk5/p25 along with Histone H1 as a substrate. Results were run on a polyacrylamide gel (Zheng et al., 2010).

When testing for new inhibitors of Cdk5 activity, Histone H1 (1 mg/ml) is generally used as a standard Cdk5 substrate. Alternatively, an NF-H peptide can be used (see above).

Roscovitine or p5 should be used as a Cdk5 inhibitor control:

Roscovitine (10μM)

p5: KEAFWDRCLSVINLMSSKMLQINA (10μM) p5 peptide was synthesized by 21^st Century Biochemicals (Marlboro, MA)

Prepare the following:

• 10 μl KB5X

• 10 μl Histone H1 (10 μg)

• 10 μl CDK5/p35 (1:50)

• 10 μl Inhibitors

• Add Water to bring up to a 40 μl volume

Mix all the reagent in the tubes by vortexing

Lastly add 10 μl of hot ATP cocktail (see Reagents and Solutions) in the radioactive room, again mix thoroughly, and spin down the contents to the bottom of the tube.

Incubate at 30°C for 60 min in an incubator.

Radioactivity can either be read on a scintillation counter (Steps 5–8; Basic Protocol 2) or using autoradiograph film (Steps 21–31; Basic Protocol 1).

REAGENTS AND SOLUTIONS

Protein lysis buffer:

• Per every 10 ml of T-PER (Thermo Scientific #78510), add 1 complete mini protease inhibitor and 1 phosphatase inhibitor tablet (Roche #11 836 153 001 and #04 906 837 001). Note: T-PER is a proprietary detergent in 25 mM bicine and 150 mM sodium chloride, but high concentrations of detergent can affect Cdk5/p39 activity, so a different lysis buffer composition may be preferred depending on the experiment (see Bankston et al., 2017). Ensure tablets of protease and phosphatase inhibitors are completely dissolved, then place on ice.

Kinase Buffer 5X (KB5X):

• Kinase Assay Buffer – 100 mM MOPS, pH 7.2, 25 mM glycerol 2-phosphate, 50 mM MgCl2, 5 mM EGTA, and 2 mM EDTA

• Reagents from Boston Bioproducts (Ashland, MA, USA):

  • MOPS Buffer (0.5 M, pH 7.4) - Products - #BB-2192
  • Beta Glycerophosphate Solution (0.2 M, Serine and Threonine Phosphatases Inhibitor) - Products - #BP-435
  • EGTA (0.5 M, pH 7.4, Autoclaved) - Products - #BM-721
  • EDTA (0.5 M, pH 7.4, Autoclaved) - Products - #BM-711

• 2M MgCl₂ (Quality Biological Inc.) Cat # 340–034-721EA

5X kinase buffer recipe: For 50 ml volume, add the following reagents:

Reagent Conc.	Required amount	Final Conc.
0.5 M MOPS Buffer	10.0 ml	100mM
0.2 M Beta Glycerophosphate	6.25 ml	25mM
2.0 M MgCl2	1.25 ml	50mM
0.5 M EGTA	0.5 ml	5mM
0.5 M EDTA	0.2 ml	2mM

Histone Mix:

• KB5X:10 μl

• CDK5 Substrate - Histone H1 (1 mg/ml) (Millipore Sigma #H5505–25mg): 10 μl

• Can make Master Mix – Multiply by n+1 samples

• Histone H1 can be reconstituted in water

Cold ATP cocktail:

• 1 ml D.W.

• 10 μl DTT 1 M (Millipore Sigma #3483–12-3)

• 2.5 μl 100 mM ATP cold (Millipore Sigma # A2383–25G)

• 10 μl Halt Protease/Phosphatase inhibitor cocktail (Thermofisher Scientific cat # 78441)

• Mix everything by vortexing.

• ATP, [γ−³²P] unpurified (PerkinElmer #NEG035C001MC)

• Aliquoting P³²:

  • Thaw the vial (−20°C) to room temperature.
  • Add 93 μl ultrapure water to 7μl of original ATP vial and vortex
  • Aliquot 10 μl of diluted P³² which contains 100 μCi of ATP for further use.

• To 200 μl of the ATP cocktail add 10 μl ATP [γ−³²P] when ready to perform Cdk5 kinase assay (adequate for 10 samples – can be scaled appropriately). Can freeze remaining cold ATP cocktail for later use.

• Note: If hot ATP reaches its half-life, then add double the amount of hot ATP to the cold ATP cocktail.

Destaining Solution:

• 300 ml Methanol (HPLC), (Fisher Chemical Fisher Scientific #A452–4)

• 50 ml Acetic acid glacial (VWR #V193–14)

• 650 ml dH₂O

COMMENTARY

Background Information

Techniques used to measure the activity of known Cdks were later applied to characterize and define the activity of Cdk5. Essentially, research with fission yeast was used to identify genes that regulate the cell cycle, leading to the discovery of cyclin-dependent kinases such as Cdk1 and Cdk2. Lew et al., (1992a and 1992b) then sought to purify homologs of cyclin dependent kinases in the brain using a synthetic peptide derived from the p34^cdc2 substrate Src. In particular, a Cdk-related protein was being searched for in the bovine brain that would possess functions other than cell cycle regulation. To characterize the newly discovered brain proline-directed protein kinase (Cdk5), a radioactive assay was used to measure phosphorylation of the Src peptide with spotting of the phosphorylated product onto a phosphocellulose paper followed by measurement of radioactivity with a liquid scintillation counter. A peptide substrate derived from another Cdk substrate, Histone H1, had even 33-fold greater phosphorylation efficiency than the Src peptide. Later, Tsai et al., 1993 developed a polyclonal antibody, PA1, to immunoprecipitate Cdk5 from various cell lines and mouse tissues and test for activity. Immunoprecipitated Cdk5 was incubated with Histone H1 and [³²P]γATP and analyzed by SDS-PAGE. Unlike the cell cycle regulator Cdk2, Cdk5 activity was highest in the mouse brain, which contains differentiated post-mitotic neurons. Cdk5 has 60% homology with cdc2 and Cdk2 (Dhavan and Tsai, 2001), and the methods initially utilized to characterize this unique member of the Cdk family have continued to be used to measure kinase activity.

Cdk5 activity is tightly regulated through interaction with its regulatory subunits p35 and p39, so the expression of these activators should be assessed to complement the results of the kinase assay. Enzymatically active Cdk5 that was isolated from bovine brain was found to be complexed with a 25 kDa regulatory subunit that was later identified as p35 (Lew et al., 1992a, Ishiguro et al., 1994, Tsai et al., 1994, and Lew et al., 1994). Following PCR cloning of the Cdk5 activator, it was determined that p35 must be further proteolytically processed to obtain p25. Degradation of p35 into the truncated p25 was speculated to occur during neuronal apoptosis, with calpain identified as the protease responsible for cleaving p35 into p10 and p25 (Kusakawa et al., 2000). Neurotoxic insults including oxidative stress, excitotoxicity, and β-amyloid (Aβ) were shown to lead to Ca²⁺ influx that then activates calpain and results in the formation of p25 (Lee et al., 2000, Nath et al., 2000, Cruz et al., 2003). Calpain cleavage of p35 to p25 cuts off the myristoylated N-terminal region that generally tethers Cdk5 activity to the plasma membrane. Overall, p35 is rapidly degraded through the proteasome pathway, with a half-life of only about 30 minutes, but p25, in contrast, has a much longer half-life. The cellular mislocalization of Cdk5/p25 along with the prolonged kinase activity may lead to phosphorylation of alternate substrates such as tau that can ultimately promote neuronal apoptosis (Dhavan and Tsai, 2001). Along with p35, a second Cdk5 activator, p39, was identified as well, that is also primarily brain specific (Tang et al., 1995). Like p35, p39 can also be cleaved by calpain to form p29, which, similar to p25, has a longer half-life than the parent full-length Cdk5 regulatory protein (Patzke and Tsai, 2002). Therefore, in conjunction with measuring kinase activity, the expression of the Cdk5 activators p35 and p39 along with their cleavage products p25 and p29 should also be studied by Western blot, particularly to determine if there is increased calpain activation along with possible neurotoxicity. Aspects of SDS-PAGE are covered in Curr. Protoc. Protein Sci. Unit 10.1 (Gallagher, 2012) and immunoblotting and immunodetection in Curr. Protoc. Cell Biol. Unit 6.2 (Ni, D., Xu, P., and Gallagher, S., 2017). If using NuPAGE™ gels, consult the NuPAGE™ technical manual.

Critical Parameters

A principal concern when running the Cdk5 assay is the use of [³²P]γATP. One should be careful to follow your institute’s guidelines for the safe handling of radioactive material. Phosphorus-32 is a beta emitter that can penetrate tissue at a range of 0.8 cm. As such, ³²P can be an external health hazard for which proper shielding and personal protective equipment should be employed. Lab coats, gloves, and safety glasses should be worn, and work with [γ−³²P] ATP should be conducted behind a 3/8-inch plexiglass shield. When using [γ−³²P] ATP, all protocol steps need to be performed within a designated radioactivity area. Monitor your workbench carefully for any spills using a Geiger-Mueller (GM) detector. When using [γ−³²P] ATP, one should both perform the kinase assay carefully to avoid spills but should also try to minimize the exposure time to radioactivity. Proper disposal of all radioactive waste should also be followed. ³²P has a half-life of about 14.3 days, so experiments should be planned to ensure that the kinase assay is performed shortly after [γ−³²P] ATP is received. We typically do not use [γ−³²P] ATP beyond two weeks after arriving in the lab.

If measuring Cdk5 activity from homogenized tissues or cell culture lysates, Cdk5 is pulled down via immunoprecipitation. The concentration of protein used (50 – 500 μg) may need to be adjusted depending on the tissue (see Basic Protocol 1, Step 6). The amount of antibody and agarose beads may also need to be fine-tuned to achieve optimal results. While immunoprecipitating Cdk5 from the protein lysates, special care must be taken to avoid aspirating the centrifuged immune complexes when performing the required washes. Appropriate lysate storage and accurate pipetting are additionally needed (see Troubleshooting). For this reason, proper Positive/Negative Controls are recommended to follow the accuracy of your technique. If measuring Cdk5 activity in lab mice, the Tgp35 transgenic mouse and the p35 knockout mouse are good to use as controls to demonstrate Cdk5 hyper- and hypoactivity respectively. However, maintaining these additional mouse strains may not be entirely feasible. An alternative to the mice can be to induce Cdk5 activity in cultured cells and use the protein lysate as a positive control. Introduction of the truncated Cdk5 activator p25 can have cytotoxic effects (Hynkova et al., 2016), but cultured cells can be transfected with an expression vector for p35 to induce Cdk5 activity using a lipid-based transfection reagent. Addgene has a pCMV-P35 (Cat # 1347) vector from Dr. Li-Huei Tsai’s Lab that contains the 955 bp human p35 coding sequence subcloned into the pCMV vector backbone. To dampen Cdk5 activity, cells can be either treated with one of the Cdk5 inhibitors listed above (Roscovitine or p5) or transfected with a dominant-negative Cdk5. D144N or K33T Cdk5 mutants impede p35 binding or interfere with the ATP-binding pocket (Su and Tsai 2011, Nikolic et al., 1996). A K33T Cdk5 vector (Addgene #1692) is available from Addgene.

Sources of recombinant Cdk5/p35 or Cdk5/p25 are readily available for purchase to identify new Cdk5 inhibitors or for examining the kinetic properties of Cdk5 mediated phosphorylation. Alternatively, Cdk5 can be isolated from an available source such as porcine brain (from a slaughterhouse), or it can be purified from the Sf9 cell baculovirus system (Minegishi et al., 2012). Derivation of recombinant GST tagged recombinant Cdk5/p35 or Cdk5/p25 is possible but the isolation of p35 or p25 can be tricky (Minegishi et al., 2012). Active Cdk5/p39 is known to play a critical role in oligodendrocyte maturation but as mentioned, Cdk5/p39 complexes are less stable than active Cdk5 complexes with p35 (Bankston et al., 2013; Bankston et al., 2017; Minegishi et al., 2012). Using p35−/− mice, we were still able to detect residual Cdk5/p39 activity. However, Cdk5/p39 complexes are sensitive to the presence of detergents, so special modifications to the above protocols may be required if studying specifically p39-mediated Cdk5 kinase activity.

Troubleshooting

Novel functions of Cdk5 are continuously being discovered in terms of both neuronal activity and non-neuronal cellular physiology, particularly with the recently identified links to cancer growth. To measure Cdk5 activity in tissues or cultured cells, thereby, requires accurate and replicable immunoprecipitation of Cdk5 that is bound to its regulatory subunits in the protein lysates. With immunoprecipitation, special care must be taken with both protein handling and with making Protein A/G immune complexes. Protein degradation in the cell lysate can subsequently affect the results of the assay. Protein lysates should be stored at −80°C for long-term storage (lysates can be stored temporarily at −20°C for no longer than 3 months). Repeated freezing and thawing should be avoided. If necessary, small aliquots of the protein lysates can be made to avoid frequent freezing and thawing. Protein lysates may also be precleared with the Protein A/G PLUS-Agarose beads (SantaCruz Biotechnology #sc-2003) if desired (Minegishi et al., 2012). Aspects of immunoprecipitation are detailed in a Current Protocols in Cell Biology chapter by Bonifacino et al., 2016. Careful washing of the agarose beads is important. Incomplete washing can affect the kinase assay. If using the Protein A/G PLUS-Agarose beads, attention must also be made to prevent aspiration of the Cdk5-bound conjugated beads. Centrifugation at too high a speed can additionally disrupt weak antibody-antigen binding, which will then impact the results of the kinase assay. One may alternatively use magnetic beads (see above) to help facilitate adequate washing of the immune complexes. Again, accurate pipetting plays a key role in ensuring good results. We generally detect Cdk5 activity by placing radiographic film directly upon the electrophoresed polyacrylamide gel, but the radioactively labeled phosphor-Histone H1 can also be transferred onto a nitrocellulose or PVDF membrane, in which case the levels of immunoprecipitated Cdk5 can be measured using immunoblotting to evaluate one’s immunoprecipitation technique (Bankston et al., 2017).

Understanding Results:

Densitometric analysis can be performed using ImageJ to quantify Cdk5 kinase activity by measuring the intensity of the radioactive signal (Figure 19). For quantification, Fiji (https://imagej.net/Fiji) needs to be downloaded– Fiji Is Just ImageJ (Schindelin et al., 2012). Scan the autoradiograph film image of Cdk5 activity along with the Coomassie blue staining of Histone H1. Open the scanned image of the autoradiograph film with Image J:

Figure 19:

Densitometric analysis of Cdk5 activity. If desired, the Cdk5 activity detected using autoradiography film can be quantified using Image J. The P³² phospho-histone H1 bands are boxed, and the pixel intensity measured.

1. Select the box tool. Ensure that the radioactive bands are horizontally aligned.

2. Draw a representative box that will outline the band. The rectangular box must also be of sufficient size to encapsulate all subsequent bands. Press 1.

3. Slide the box over to cover the adjacent band, then press 2. Continue until all bands are accounted for. Then press 3. A plot of pixel intensity should appear.

4. Select the line tool.

5. Use the line tool to trim the edges of each curve.

6. Choose the wand tool and then select each adjusted pixel plot. The area under the curve should appear. Copy these values into Excel.

7. Statistical analysis can then be performed using GraphPad Prism.

After finishing with the autoradiography film, measure the pixel intensity of the Coomassie-stained Histone H1 bands. One should calculate the value of ³²P radioactivity / Histone H1 in your analysis. Densitometry requires that the signal be in the linear range of detection. Nonlinearity and errors in normalization can occur with autoradiography film if bands are over-exposed and saturated (Butler et al. 2019). Generally, we use a positive control just to ensure that the assay works overall, but an appropriate dilution of the recombinant kinase is needed if used for quantification purposes. If desired, densitometric analysis can be performed using ImageJ for Western blot as well except using the intensity of the chemiluminescent signal of Cdk5 (or its activators p35 and p39) over the protein loading control.

Results, whether CPM from the scintillation counter or measurements derived from densitometric analysis, should then be entered into GraphPad Prism or a preferred graphing program. Statistical significance can be determined, as GraphPad comes with built-in statistical tools to run a Student’s t-test or ANOVA.

Time Considerations

Depending on the method used to detect radioactivity, the Cdk5 assay can take between 1 day to a full week. In general, it takes approximately one full day to get results using a scintillation counter. The assay should be started in the morning, where the kinase reaction itself takes about an hour. After spotting onto the P81 phosphocellulose squares, washing, and air drying, the amount of radioactivity can then be measured on a scintillation counter. Measuring the radioactivity takes a few hours and can be left overnight. If Cdk5 needs to be pulled down from a protein lysate, the immunoprecipitation step generally requires an overnight incubation to develop Protein A/G immune complexes. If using film to detect kinase activity, time is needed to set up the experiment, run the kinase reaction (~60 min), then load the samples on a gel (~1 ½ hours). If needed, the finished kinase reaction can be frozen at −30°C and electrophoresed on a later day. The polyacrylamide gel needs to be stained with Coomassie Blue and destained overnight. Multiple exposures may then be needed to get the right image with film, generally starting with an overnight exposure. The entire process using the SDS-PAGE method takes about a week.

Running a Western blot to detect the Cdk5 activators generally takes between 2 – 4 days. Gel electrophoresis and transfer of separated proteins onto a membrane can take 4 hours or less depending on the voltage used and the type of protein transfer (dry or wet blotting). Primary antibodies are generally incubated overnight at 4°C. About a couple of hours is then needed the next day for the secondary antibody and subsequent chemiluminescent detection. Another overnight incubation is needed for the protein loading control (i.e., GAPDH, Actin, or α-Tubulin). Membranes can be stripped again following the detection of the loading control to then test another antibody (the following scheme can be done for example: detect p35 [rabbit polyclonal antibody], strip the membrane, detect beta actin [mouse monoclonal], strip the membrane again, then examine Cdk5 [rabbit polyclonal antibody]).

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.