Kinase-Catalyzed Crosslinking and Immunoprecipitation (K-CLIP) to Explore Kinase-Substrate Pairs

Abstract

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using Kinase-catalyzed Crosslinking and Immunoprecipitation (K-CLIP). K-CLIP utilizes a photocrosslinking γ-phosphoryl modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology.

Basic Protocol 1:

Kinase-catalyzed crosslinking of lysates

Basic Protocol 2:

Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP)

INTRODUCTION:

Phosphorylation is a key protein post-translational modification that controls interactions, conformations, and/or functions of the modified protein (Wang and Cole, 2014). By governing protein activity, phosphorylation regulates a myriad of biological processes, including cell signaling (Krupa et al., 2004). As a critical mediator of cellular events, protein phosphorylation is highly regulated, and diseases, such as cancer and Alzheimer’s disease, develop when phosphorylation events become dysregulated (Lu, 2004). Protein kinases catalyze phosphorylation by transferring the γ-phosphoryl of adenosine 5’-triphosphate (ATP, Figure 1A) onto a serine, threonine, or tyrosine of substrates to generate phosphoproteins (Figure 1A). With a significant role in disease, kinases have been effective targets for pharmaceutical drug development, with many kinase inhibitors in clinical use (Gross et al., 2015). To thoroughly characterize the role kinases play in cellular events and disease states, identification of upstream kinases of a phosphoprotein and downstream substrates of a kinase is essential. Currently, the characterization of kinase-substrate pairs is often difficult due to the weak and transient interaction between kinases and their substrates (Shaffer and Adams, 1999). Development of chemical tools to identify the kinases that phosphorylate a specific phosphoprotein is vital for deciphering the intricate network of protein-protein interactions in cellular processes.

Figure 1. Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).

(A) General scheme for phosphorylation of a protein substrate by a kinase using ATP as the cosubstrate. (B) Chemical structure of photocrosslinking ATP analog, ATP-arylazide (ATP-ArN₃). (C) Kinase-catalyzed labeling with ATP-ArN₃ covalently attaches the arylazide photocrosslinker to the substrate. (D) UV irradiation results in the formation of crosslinked kinase-substrate complexes through the arylazide group. (E) Immunoprecipitation (IP) of the substrate will isolate substrate-bound complexes, which then can be characterized by Western blotting after SDS-PAGE separation or LC-MS/MS analysis. (F) Due to the transient nature of kinase-substrate interactions, a significant amount of substrate will diffuse away from the kinase prior to UV irradiation and crosslinking. (G) Upon UV irradiation after diffusion, the arylazide group will crosslink the substrate to nearby proteins, which will include associated proteins (Garre et al., 2018).

To create effective chemical tools to study cellular events, γ-phosphoryl modified ATP analogs have been established to study kinases, substrates, and protein phosphorylation. A variety of γ-phosphoryl modifications, such as biotin (Green and Pflum, 2007), arylazide (Suwal and Pflum, 2010), benzophenone (Garre et al., 2014) and methylacrylamide (Fouda et al., 2021), have been attached to ATP. These ATP analogs are accepted by kinases as cosubstrates whereby the modified γ-phosphoryl is transferred onto the hydroxyl of serine, threonine, or tyrosine amino acids of substrate proteins (Figure 1C), which is known as kinase-catalyzed labeling (Senevirathne et al., 2016). After labeling of the phosphoprotein, a variety of methods have been developed to study the interactions between a kinase and substrate to advance the current understanding of cellular events (Dedigama-Arachchige and Pflum, 2016; Embogama and Pflum, 2017; Garre et al., 2018; Ramanayake-Mudiyanselage et al., 2021).

This protocol focuses on one specific kinase-catalyzed labeling reaction involving covalent crosslinking to study substrate-kinase interactions (Suwal and Pflum, 2010). Specifically, kinase-catalyzed crosslinking covalently joins kinases and substrates to overcome their normally weak and transient interaction, which facilitates subsequent enrichment and identification. Although multiple crosslinking ATP analogs have been developed for kinase-catalyzed crosslinking (Fouda et al., 2021; Garre et al., 2014; Suwal and Pflum, 2010), we focus here on the photocrosslinking ATP-arylazide analog (ATP-ArN₃, Figure 1B), given its prior use towards kinase-substrate identification (Dedigama-Arachchige and Pflum, 2016; Garre et al., 2018). In kinase-catalyzed crosslinking with ATP-ArN₃, the photoreactive arylazide group is transferred to the substrate after kinase-catalyzed labeling (Figure 1C). Upon UV irradiation, a highly reactive nitrene species is generated from the arylazide (Wilson et al., 1975) that can covalently attach to the backbone or amino acid side chains of the kinase (Figure 1D). The kinase-catalyzed crosslinking reaction results in a stable kinase-substrate complex for subsequent analysis.

To use kinase-catalyzed crosslinking for kinase-substrate monitoring or discovery, an enrichment step is necessary to isolate the substrate and kinase of interest after crosslinking. In this protocol, the Kinase-catalyzed Crosslinking and Immunoprecipitation (K-CLIP) method (Garre et al., 2018) is highlighted where a primary antibody is used in an immunoprecipitation step to enrich the crosslinked kinase-substrate complex (Figure 1E). Depending on whether the immunoprecipitation enrichment step uses a primary antibody that targets a substrate or kinase of interest, the K-CLIP method can identify the upstream kinases of a phosphoprotein substrate or the downstream substrates of a kinase. Given our prior published work applying K-CLIP to the identification of kinases of a target substrate phosphoprotein (Garre et al., 2018), this protocol focuses on substrate immunoprecipitation (Figure 1E) and the analysis of upstream kinases of a phosphoprotein. We note that application of K-CLIP to the identification of substrates of a target kinase is currently ongoing in the Pflum lab. Finally, to analyze the enriched crosslinked complexes, sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) separation and Western blot analysis can be used to probe for the presence of suspected kinase-substrate pairs, which provides a helpful lysate-based confirmation tool to complement in vitro kinase assays with recombinant proteins. Alternatively, unanticipated kinase-substrate pairs in the enriched crosslinked complexes can be identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), which makes K-CLIP a powerful discovery tool. Overall, K-CLIP is a versatile method capable of monitoring the active phosphorylation of any kinase or substrate of interest.

In prior work, K-CLIP was used to identify kinase(s) of the p53 protein (Garre et al., 2018). Because p53 is robustly phosphorylated by many known kinases (Kruse and Gu, 2009; Maclaine and Hupp, 2009), p53 was an ideal substrate model to establish the method. K-CLIP combined with LC-MS/MS analysis successfully identified two known (DNA-PK and PKR) and one unknown (MRCKβ) kinases of p53 (Garre et al., 2018). In addition, K-CLIP followed by SDS-PAGE separation and Western blot analysis confirmed known kinase-substrate pairs (Garre et al., 2018). The p53 model study established the K-CLIP method to both discover and validate the kinases of a target substrate in a complex cellular mixture.

Interestingly, the p53 K-CLIP study identified many proteins in addition to kinases, including known associated proteins of p53. Based on the data, we rationalized that associated proteins are crosslinked and identified by K-CLIP due to the transient interaction between kinase and substrate that allows the substrate to diffuse away from the kinase before UV irradiation (Figure 1F). In this case, diffusion of the modified substrate results in crosslinking to associated proteins that are near the substrate and kinase (Figure 1G). In fact, other work using kinase-catalyzed crosslinking and a substrate peptide similarly observed both kinases and associated proteins after LC-MS/MS analysis (Dedigama-Arachchige and Pflum, 2016). In total, the data documents that K-CLIP is useful to study the associated proteins of kinases and substrates during the phosphorylation event, in addition to kinase-substrate pairs.

To facilitate future use of K-CLIP for both confirmation and discovery of kinase-substrate pairs and associated proteins, detailed protocols are provided here for kinase-catalyzed crosslinking reactions and the K-CLIP method using ATP-ArN₃. Given the prior published p53 example, the protocols are focused on phosphoprotein substrate enrichment to identify kinases, with specific details for p53 as a model system. First, Protocol 1 is provided to observe kinase-catalyzed crosslinking in complex lysate mixtures using SDS-PAGE separation and Western blot analysis, with p53 as the example protocol. For application of K-CLIP to new phosphoprotein substrates, Protocol 1 provides a necessary first step to ensure that ATP-ArN₃ and kinase-catalyzed crosslinking are compatible with the target substrate. Second, Protocol 2 describes the K-CLIP method using substrate immunoprecipitation enrichment steps, with p53 as the model system. Protocol 2 is suitable for either confirmation of suspected kinase-substrate pairs using SDS-PAGE separation and Western blot analysis or discovery of unanticipated kinases and associated proteins using LC-MS/MS analysis. In total, the two protocols will assist in applying K-CLIP to any substrate phosphoprotein of interest to either confirm or discover kinases and associated proteins, which has the anticipated outcome of improving the current understanding of phosphorylation-mediated cell signaling and disease states.

BASIC PROTOCOL 1

Basic protocol title:

Kinase-catalyzed crosslinking of lysates

Introductory paragraph:

As a prerequisite of K-CLIP, the compatibility of kinase-catalyzed crosslinking and ATP-arylazide with the kinase-substrate pair of interest must be verified. Basic Protocol 1 outlines kinase-catalyzed crosslinking with Western blot analysis to observe high molecular weight crosslinked complexes, which confirms crosslinking of cellular proteins with the substrate of interest within the desired lysate. When applying Protocol 1 to a target kinase-substrate pair, an important consideration is the selection of the lysate containing the substrate and kinases of interest. The versatility of the K-CLIP method allows for the use of almost any lysate or protein mixture, such as mammalian cell, bacterial, or yeast lysates, or tissue homogenates. Please see the Critical Parameters section for a discussion on lysate selection.

For Basic Protocol 1, several control reactions should be included, in addition to the crosslinking reaction. Where the crosslinking reaction contains lysate and ATP-arylazide with UV irradiation (Table 1, crosslinking reaction), a critical negative control reaction should be performed containing lysate and ATP-arylazide without UV light (Table 1, negative control #1) to show the UV dependence of crosslinking. Other optional negative control samples include reactions incubated without ATP-arylazide (Table 1, optional negative control #2), with ATP in place of ATP-arylazide (Table 1, optional negative control #3), or with kinase inhibitor-treated lysates (Table 1, optional negative control #4), which establish the ATP analog and kinase activity dependence of crosslinking. The reactions are typically incubated for 2 hours at 31 °C, but the reaction time may vary depending on the lysates and proteins involved.

Table 1.

Suggested general kinase-catalyzed crosslinking reaction conditions

	Crosslinking Reaction	Negative Control #1	Optional Negative Control #2	Optional Negative Control #3	Optional Negative Control #4
Lysate	+	+	+	+	-
Inhibitor-treated Lysate	-	-	-	-	+
ATP	-	-	-	+	-
ATP-arylazide	+	+	-	-	+
UV	+	-	+	+	+

In Protocol 1, the samples that undergo kinase-catalyzed crosslinking are separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and visualized by Western blotting. For Western blotting, the primary antibody should be specific for the substrate of interest, and the secondary antibody can be conjugated to either a fluorophore or horseradish peroxidase (HRP), depending on the level of sensitivity desired. Crosslinking is validated by observing high molecular weight protein bands in the crosslinking reaction, which are reduced in the negative control reactions (Table 1). Depending on the efficiency of crosslinking, the higher molecular weight complexes might appear smeared without distinct bandings. Please see the Anticipated results section for example gel images.

Materials:

• ATP-arylazide: synthesized from Suwal and Pflum, 2010 (see Reagents and Solutions)

• Cell lysates stock solution: HEK293 or other user provided lysate, prepared in HEPES lysis buffer (see Reagents and Solutions).

• 10X Kinase reaction buffer: see Reagents and Solutions

• β-mercaptoethanol: Sigma-Aldrich cat. No. M6250

• 4X Gel Loading Buffer for SDS-PAGE: 4X Laemmli Protein sample buffer, Bio Rad cat. No. 1610747. Prepared as directed by the manufacturer.

• Phosphate Buffered Saline with Tween (PBST): see Reagents and Solutions

• Blocking Solution: 5% BSA or nonfat milk in 1X PBST.

• Primary Antibody: Anti-p53 (Santa Cruz, cat. No. sc-126)

• Secondary Antibody: Goat Anti-mouse IgG H&L (HRP) (Abcam, cat. No. ab97040)

• 1.5 mL Centrifuge Tubes: Fisherbrand (cat. No. 05–408-129)

• Benchtop Micro Centrifuge: FisherScientific accuSpin Micro 17

• UV Lamp: 3UV lamp 254/302/365nm, UVP

• Thermomixer: Multi-Therm Heat-shake, Benchmark Scientific

• 10–16% SDS-Polyacrylamide gel: Buy commercial gels or make gels as previously published (He, 2011).

• Electrophoresis apparatus: Mini-Protean Tetra Cell (BioRad)

• PVDF membrane: Immobilon P^SQ-Millipore, cat. No. IPVH00010

• Electrotransfer apparatus: Mini-Transblot Module (BioRad)

• Rocking Platform: VWR 200 Rocking Platform

• Imaging Instrument: Alpha View-Fluorochem Q (ProteinSimple) or GE FLA 9500 imager (Typhoon).

Optional Materials

• Adenosine 5’-triphosphate: MP Biomedicals Inc, cat. No. ICN15026605

• Enhanced Chemiluminescence (ECL) Substrate for HRP Visualization: SuperSignal West Dura Extended Duration Substrate (ThermoFisher cat. No. 34075)

Protocol steps with step annotations:

Kinase-catalyzed crosslinking of cell lysates

1. Label centrifuge tubes with reaction numbers (Table 2).

2. Add enough 10X kinase buffer to obtain a final concentration of 1X into the labeled centrifuge tube(s) and dilute with enough deionized, purified water such that all samples have the same final volume. In this example with p53, add 2 μL of 10X kinase buffer and 10 μL of water, to obtain a final volume of 20 μL.

   Typically use a HEPES buffer. Buffers containing primary amines, such as Tris, are incompatible due to competition with crosslinking.

   The final volume for the kinase reactions may vary depending on the initial concentration of proteins in the lysates (see step 3 for more information).

   As shown in Table 1, inclusion of controls is highly recommended. In this example protocol with p53, the negative controls include omitting UV irradiation (Table 2, Negative Control #2) and replacing ATP-arylazide with ATP (Table 2, Negative Control #2), which are the minimum controls recommended for Basic Protocol 1.

3. Thaw the lysates on ice, and once thawed, add to the tube(s). In the p53 example, add 6 μL of 21 mg/mL lysate (125 μg total protein) to the tube(s).

   Lyse cells in a buffer that does not contain free amines, such as HEPES, to avoid competition with crosslinking.

   To avoid degradation of lysate proteins from freeze thaw cycles, store as single use aliquots. Pipetting must be performed on ice or in a cold room, with prechilled centrifuge tube(s).

   For this protocol, 125 μg of total protein in the lysate was used, but crosslinking may require more or less lysates depending on the abundance of the substrate of interest. Most kinase-catalyzed crosslinking reactions use 30–800 μg of lysate. Testing multiple lysate amounts prior to crosslinking experiments is recommended to consider substrate abundance.

   Lysate stock concentrations used previously have ranged from 5 mg/mL to 45 mg/mL. The concentration of lysate will impact the total volume of the reaction. Depending on the lysate concentration, the total volume of lysates can range from 5–190 μL, which results in the total reaction volume ranging from 15–250 μL. Using a more concentrated lysate is recommended, when possible, to have a more concentrated total reaction volume, which can promote more interactions between the kinases and substrates.

4. Thaw ATP-arylazide (and optional ATP aliquots) on ice. Initiate the kinase reaction(s) by adding ATP-arylazide (or ATP) to a final concentration of 5 mM. In the p53 example, add 2 μL of 50 mM ATP-arylazide (or ATP). To ensure all reagents are combined, spin down reactions for 5–10 seconds.

   To avoid degradation of ATP and the ATP analogs from freeze thaw cycles, store as single use aliquots. Pipetting must be performed on ice or in a cold room, with prechilled centrifuge tube(s).

   The final concentration of ATP-arylazide in the kinase reactions might need to be optimized. The concentration of ATP-arylazide used previously varied from 2 mM to 10 mM.

5. Incubate the reaction sample(s) immediately at 31°C with (Figure 2A and 2B) or without (Figure 2C) UV irradiation (365 nm) with shaking at 300 rpm for 2 hours using a thermomixer.

   To promote efficient crosslinking, the UV lamp should be as close to the sample tubes as possible to activate arylazide (see Figure 2B). The distance of the UV lamp from the tube is critical to induce crosslinking, which might need to be optimized. The temperature of the reaction might also need optimizing.

Table 2.

Kinase-catalyzed crosslinking reaction conditions for p53^*

	Negative Control #1	Negative Control #2	Crosslinking Reaction
HEK293 lysate (125 μg)	6 μL	6 μL	6 μL
Kinase Buffer (1X)	2 μL	2 μL	2 μL
ATP (5 mM)	2 μL	-	-
ATP-arylazide (5 mM)	-	2 μL	2 μL
Water	10 μL	10 μL	10 μL
UV	+	-	+

^*In the example experiment for Protocol 1, p53 was used as the substrate of interest and the stock concentrations were 21 mg/mL HEK293 lysate, 50 mM ATP-arylazide, and 50 mM ATP.

Figure 2. Thermomixer setup with UV lamp for crosslinking.

(A) The general setup of the UV irradiation area is shown, which includes the thermomixer, UV lamp, and the support stands. (B) The samples to be UV irradiated are placed open in the thermomixer with a UV lamp placed 0.2–0.3 cm above the top of the centrifuge tubes, which is a total of 1 cm from the top of the thermomixer platform. The centrifuge tubes used are about 4 cm in length, which results in the UV light being a total of 3.8–4 cm away from the sample reaction. (C) Samples that do not require UV irradiation are covered with foil and placed in the thermomixer as far away as possible from the UV lamp.

Safety note: UV light is harmful; avoid direct contact of the light to the eyes and skin

SDS-PAGE and Western blotting

1. After the reaction incubation, add 4X Gel Loading Dye (GLD) to a final concentration of 1X to the kinase reactions and incubate the sample(s) at 95 °C for 5 minutes to heat denature the proteins. For this p53 example protocol, add 6.6 μL of 4X GLD to each sample.

2. Prepare SDS-polyacrylamide gels following established protocols (He, 2011) or use commercial gels. For the p53 example, which is 53 kDa, use a 10% gel.

   The percentage gel used depends on the size of the protein of interest. Use a higher percentage gel (i.e., 16%) to observe lower molecular weight proteins (MW<30 kDa) and a lower percentage gel (i.e., 8%) for higher molecular weight proteins (MW>150 kDa). The ideal gel percentage chosen should allow for visualization of the substrate of interest as well as higher molecular weight complexes.

3. Load the gel with the reaction samples and separate the proteins using 110 V for 15 minutes (or until dye front is through the stacking layer) and then increase voltage to 180 V for about 45 minutes (or until the dye front runs off the gel). SDS-PAGE protocols are published elsewhere (Manns, 2011).

   The run time for separation depends on the size of the substrate. For larger protein substrates, the gel may be run longer to ensure visualization of higher molecular weight crosslinked complexes. For smaller protein substrates, the gel may be run shorter than 1 hour to ensure the substrate of interest remains on the gel.

4. Transfer the separated proteins in the gel onto a PVDF membrane at 90 V for 2 hours using an electroblotting apparatus (Goldman et al., 2015).

5. After protein transfer, block the membrane with blocking solution. In the p53 example, block the membrane with 10 mL of 5% (w/v) nonfat milk in 1X PBST for 1 hr at rt.

   The blocking step may need to be optimized to minimize background. Blocking may also be performed using bovine serum albumin (BSA) or a blocking solution such as SuperBlock (ThermoFisher).

6. Remove the blocking buffer and wash the membrane 3 times with 30 mL of 1X PBST for 3 minutes with rocking.

7. Incubate the membrane with the manufacturer recommended dilution of primary antibody in 5% (w/v) BSA or nonfat milk in 1X PBST for 1 hr or overnight at 4 °C. For the p53 example, add 10 mL of a 1:1000 dilution of p53 primary antibody in 5% (w/v) BSA in 1X PBST to the membrane and incubate overnight at 4 °C.

   The primary antibody, dilutions, and incubation time should be used according to manufacturers’ recommendation or optimized to obtain the best quality images.

8. After primary antibody incubation, remove the solution and wash the membrane 3 times with 30 mL of 1X PBST for 3 minutes with rocking.

9. Incubate the membrane with the manufacturers’ recommended dilution of HRP- or fluorophore-conjugated secondary antibody in 5% (w/v) nonfat milk or BSA in 1X PBST for 1 hr at room temperature. For p53, incubate the membrane with 10 mL of 1:10,000 dilution of an HRP-conjugated secondary antibody in 5% (w/v) nonfat milk in 1X PBST for 1 hour at rt.

   The secondary antibody and dilutions used should be optimized to obtain the best quality images.

10. Remove the secondary antibody and wash the membrane again 3 times with 30 mL of 1X PBST for 3 minutes with rocking.

11. After washing, directly visualize the fluorophore-labeled secondary antibody or incubate the membrane with 1 mL ECL Plus reagent when using secondary HRP antibody, according to manufacturers’ protocol (TECH, 2018). For the p53 example, incubate with 1 mL (500 μL luminol/enhancer + 500 μL stable peroxide buffer) of ECL Plus reagent. Visualize the membrane using a chemiluminescent scanner.

    The gel image for the p53 kinase-catalyzed crosslinking example is shown in Figure 3.

Figure 3. Kinase-catalyzed crosslinking of endogenous p53 in HEK293 cell lysates.

HEK293 cell lysates (125 ug; all lanes) were incubated with ATP (lane 2) or ATP-arylazide (lanes 3 and 4) in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of UV irradiation. Proteins were separated by 10% SDS-PAGE and electrotransferred to a PVDF membrane. Western blotting with a p53 primary antibody (α-p53) visualized p53 (arrow) and p53-crosslinked complexes (bracket). The molecular weight marker bands are indicated in kDa to the left of the gel image.

BASIC PROTOCOL 2:

Basic protocol title:

Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP)

Introductory paragraph:

After observing crosslinked complexes with the desired substrate and lysate mixture (Basic Protocol 1), the next step is to isolate the crosslinked complexes to identify the kinase-substrate or substrate-associated protein pairs either by Western blot or LC-MS/MS analysis. Because kinase-catalyzed crosslinking will covalently conjugate all kinases and substrates in a lysate mixture, the immunoprecipitation enrichment step of K-CLIP (Figure 1E) is required to identify only the proteins that are crosslinked to the substrate of interest. We provide in this protocol two methods to perform the immunoprecipitation enrichment step, which differ in how the substrate-antibody-agarose bead conjugate is generated. Method A involves incubating the primary antibody with agarose beads before adding lysates containing the crosslinked substrate, whereas Method B involves incubating the primary antibody with crosslinked lysates prior to addition of the agarose bead. Method A is more commonly used for immunoprecipitation. However, in cases where 1) the abundance of the substrate of interest is low, 2) the binding affinity of the primary antibody for the substrate of interest is weak, and/or 3) the binding kinetics of the primary antibody for the substrate of interest is slow, Method B is recommended (Bates; TIP, 2009). Alternatively, other enrichment methods can be used that are compatible with the substrate of interest. Please see the Critical Parameters section for more information on alternative enrichment methods.

Basic Protocol 2 focuses on the immunoprecipitation enrichment step after kinase-catalyzed crosslinking (Basic Protocol 1). The bound proteins after immunoprecipitation can subsequently be separated by SDS-PAGE, electrotransferred to a PVDF membrane, and analyzed by Western blot analysis, as previously described (Basic Protocol 1). In addition to probing the target substrate, antibodies to a suspected kinase or associated protein can also be tested, if interested. After confirmation of high molecular weight complexes in the enriched sample by Western blot analysis, the K-CLIP experiment can be repeated for proteomics analysis, as published elsewhere (Garre et al., 2018; Shevchenko et al., 1996).

Additional Materials (see also Basic Protocol 1):

• Protein A/G plus-agarose beads: Santa Cruz Biotechnology, cat. No. sc-2003

• Tris Buffered Saline (TBS) (see Reagents and Solutions)

• Primary Antibody: Anti-p53 (Santa Cruz, cat. No. sc-126)

• HEPES Lysis buffer (see Reagents and Solutions)

• Lysis buffer (see Reagents and Solutions)

• 2X Gel Loading Buffer for SDS-PAGE: Dilute 4X Laemmli Protein sample buffer (Bio Rad cat. No. 1610747) to make a 2X buffer. Add β-mercaptoethanol reducing agent, as suggested by manufacturer.

• Rotisserie Tube Rotator: Mini LabRoller Rotator (Labnet)

Optional Materials

• Light chain specific secondary antibody: goat anti-mouse HRP light chain specific (Airgo Biolaboratories, cat. No. ARG21551). Note: Use a light chain specific secondary antibody after immunoprecipitation to visualize the protein bands if the substrate of interest has a similar molecular weight as the heavy chain (50 kDa).

Protocol steps with step annotations:

Kinase-catalyzed crosslinking with lysates

• 1Use Protocol 1 to initially perform kinase-catalyzed crosslinking of cell lysates. For Western blot analysis, use 500 μg of lysate in the kinase-catalyzed crosslinking reactions (Table 3). For subsequent proteomics analysis, 1 mg or more lysate should be used. In the case of Western blot analysis, an amount of lysate sufficient for visualizing crosslinking is needed. A larger amount of lysate is typically used for proteomics analysis to ensure sufficient peptide levels after trypsin digestion for LC/MS-MS. In this example, only K-CLIP for Western blot analysis is described here.

Table 3.

K-CLIP reaction conditions for p53 and subsequent Western blot analysis ^*

	Negative Control #1	Negative Control #2	Crosslinking Reaction
HEK293 lysate (500 μg)	13.5 μL	13.5 μL	13.5 μL
Kinase Buffer (1X)	4 μL	4 μL	4 μL
ATP (5 mM)	4 μL	-	-
ATP-arylazide (5 mM)	-	4 μL	4 μL
Water	18.5 μL	18.5 μL	18.5 μL
UV	+	-	+

^*For Basic Protocol 2, the stock concentrations are 37 mg/mL HEK293 lysate, 50 mM ATP-arylazide, and 50 mM ATP. The HEK293 lysate contains kinases and the p53 substrate endogenously.

Immunoprecipitation

• 2Inclusion of controls are recommended for immunoprecipitation (IP, see Table 4). In addition to the reaction controls from Protocol 1 (Table 4), a necessary negative control contains the crosslinked lysate and beads, but no antibody, which will identify nonspecifically bead-bound proteins (Table 4, IP Negative Control #1). Another helpful negative control is an antibody-agarose bead sample to distinguish the bands of the antibody from the substrate of interest (Table 4, IP Optional Negative Control #2), which is particularly helpful if the target substrate has a molecular weight similar to the light (25 kDa) or heavy (50 kDa) chain. For the antibody-agarose bead control, the primary antibody and agarose beads, but no lysates, are included.

Table 4.

Setup for Immunoprecipitation of p53 samples^*

	Negative Control #1	Negative Control #2	Crosslinking Reaction	IP Negative Control #1	Optional IP Negative Control #2
Basic Protocol 1 Kinase reaction samples	40 μL	40 μL	40 μL	40 μL	-
p53 Antibody (1:1000)	10 μL	10 μL	10 μL	-	10 μL
Agarose beads	20 μL	20 μL	20 μL	20 μL	20 μL

^*For the p53 example, method A was used for immunoprecipitation.

Preparation of protein A/G plus agarose beads

• 3Label centrifuge tubes with reaction numbers (Table 4).
• 4Add 20 μL of bead slurry to the labeled centrifuge tubes.

  Vortex the stock of beads to make a homogenous mixture before dispensing and use a pipette tip that has the end cut to make a wider opening that avoids damaging the beads. Keep all bead-containing tubes on ice to avoid degradation throughout the immunoprecipitation procedure.

• 5Wash the beads by adding 500 μL of 1X TBS, inverting 3 times, and centrifuging for 1 minute at 5000 rcf at 4 °C to collect the beads. Gently remove and discard the 1X TBS wash solution using a pipette.

  Remove sample tubes carefully from the centrifuge to avoid disrupting the bead pellet.

• 6Repeat step 3 once more to wash the beads a second time. Use the washed beads immediately.

Immunoprecipitation Method A

• 7After washing the beads twice, add primary antibody for the target protein. For this example, add 10 μL of p53 primary antibody and dilute to 500 μL with HEPES lysis buffer.

  Typically, 1–10 μL (0.2–2 μg) of primary antibody is used for immunoprecipitation. The antibody manufacturer will often give advice on the optimal antibody amount for immunoprecipitation. However, optimization of antibody quantity might be needed.

• 8Rock the bead-antibody mixture for 1 hour to overnight at 4 °C with end-over end rotation. For the p53 example, incubate the antibody with the beads for 1 hr.
• 9After incubation of the antibody and beads, centrifuge the mixture for 1 minute at 5000 rcf at 4 °C. Remove and discard the HEPES lysis buffer carefully using a pipette to avoid disrupting the beads.
• 10Add 500 μL of HEPES lysis buffer, invert beads 3 times, centrifuge at 5000 rcf for 1 min, and carefully remove the buffer without disrupting the beads to remove unbound antibody.
• 11Repeat step 4 a total of 3 times to wash the beads.
• 12After the third wash, store the washed beads on ice until the crosslinking reactions from Protocol 1 are complete.

  The beads should not be stored for a long period of time while waiting for the crosslinking reactions. We suggest that the beads are washed when there is about 10 minutes left of the 2 hour crosslinking reactions.

• 13Add the crosslinking reaction(s) to the separate tubes of washed beads and dilute with HEPES lysis buffer to a total volume of 1 mL.
• 14Rock the reaction(s) overnight at 4 °C or for manufacturers’ suggested amount of time with end over end rotations. For the p53 example, incubate the samples overnight at 4 °C.

  The incubation time with the crosslinking reactions and the antibody-conjugated agarose beads should be optimized for the antibody being used.

• 15After incubation, centrifuge the reactions at 5000 rcf at 4 °C for 1 minute and carefully remove the 1 mL supernatant without disrupting the beads.
• 16Add 1 mL of lysis buffer, invert tubes 3 times, and centrifuge at 5000 rcf at 4 °C for 1 minute. Remove and discard the lysis buffer.
• 17Repeat step 10 a total of 3 times to wash the beads. Proceed to preparation of samples for gel analysis section.

Immunoprecipitation Method B

• 18To the crosslinking reaction(s) from Protocol 1, add the manufacturers’ recommended amount of primary antibody for immunoprecipitation and rock overnight at 4 °C or for manufacturers’ recommended time with end over end rotations.

  The dilution of primary antibody and the amount of time to incubate the antibody with the reaction samples may need to be optimized.

• 19Prepare washed agarose beads as described previously (see Preparation of protein A/G plus agarose beads).
• 20Add each antibody-containing reaction mixture to a tube of washed beads, dilute up to 1 mL with HEPES lysis buffer, and rock at 4 °C with end over end rotations for 3 hours.

  The incubation time of the kinase-antibody mixture with the washed agarose beads might also need to be optimized for the chosen substrate of interest.

• 21After incubation of the antibody-containing reaction samples with the beads, centrifuge at 5000 rcf at 4 °C for 1 minute, and then carefully remove and discard the supernatant without disrupting the beads.
• 22Add 1 mL of lysis buffer to the beads, invert 3 times, centrifuge at 5000 rcf for 1 minute at 4 °C, and then remove and discard the lysis buffer.
• 23Repeat step 5 a total of 3 times to wash the beads. Proceed to preparation of samples for gel analysis section.

Preparation of Samples for Gel Analysis

• 24To the bound and washed beads from Method A or B, add 20 μL of 2X gel loading dye to each tube.
• 25Heat the bound beads at 95 °C for 5 minutes to elute and denature proteins from the beads.
• 26Prepare SDS-polyacrylamide gels following established protocols (He, 2011) or use commercial gels. For the p53 example, a 10% gel was used.
• 27Centrifuge the samples at 5000 rcf for 1 minute to collect the samples.
• 28Run SDS-PAGE and Western blot analysis as described in Basic Protocol 1.

  The gel image for the p53 K-CLIP example is shown in Figure 4.

Figure 4. K-CLIP with endogenous p53 in HEK293 cell lysates.

HEK293 cell lysates were incubated with ATP (lane 1) or ATP-arylazide (lane 2 and 3) in the presence (lane 1 and 3) or absence (lane 2) of UV irradiation. After immunoprecipitation of p53 from the lysates, bound proteins were separated by 10% SDS-PAGE followed by transfer to PVDF membrane. Western blotting with a p53 primary antibody (α-p53) visualized p53 (arrow) and p53-crosslinked complexes (bracket). A sample containing only the p53 primary antibody and Protein A/G agarose beads used for immunoprecipitation was also included (lane 4). Molecular weight marker bands are indicated in kDa to the left of the gel image.

REAGENTS AND SOLUTIONS:

Use deionized, purified water for all steps and recipes.

ATP-arylazide Stock Solution

• Dissolve ATP-arylazide powder in 100 μL of 100 mM HEPES, pH=7.4. Determine the absorbance via UV-Vis spectrophotometer and calculate concentration using beers law (A=εbc) where A = the absorbance at 254 nm, ε = 15.4 × 10³ M⁻¹ cm⁻¹, b = path length (cm), and c = concentration of ATP-arylazide. The concentration after resuspending solid ATP-arylazide ranges from 30–200 mM, which may need to be diluted before use in kinase reactions.

• Make 5 or 10 μL single use aliquots and store at −80 °C. Avoid freeze-thaws to prevent degradation.

• Shelf life: up to 1 year as a dry powder in −80 °C and up to 6–8 months after dissolved in buffer at −80 °C.

• Purity: the ATP-arylazide should show minimal degradation by TLC analysis (silica; 3:1.5:0.5 isopropanol:ammonium hydroxide:water; R_f=0.4)

HEPES Lysis Buffer

• 50 mM HEPES, 150 mM NaCl, 10% (w/v) glycerol, and 0.5% (w/v) Triton X-100.

• Adjust the pH to 8.0 using HCl and/or NaOH.

• Store at 4 °C for up to 6 months

Lysis Buffer

• 50 mM Tris-HCl, 150 mM NaCl, 10% (w/v) glycerol, and 0.5% (w/v) Triton X-100.

• Adjust the pH to 8.0 using NaOH and/or HCl.

• Store at 4 °C for up to 6 months

Phosphate Buffered Saline with Tween (PBST, 1X)

• 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄.

• Adjust pH to 7.4 with HCl and/or NaOH.

• Add 0.1% (v/v) Tween-20 from commercial stock

• Store at room temperature for up to 1 year

Reaction Buffer (10X)

• 250 mM HEPES, 500 mM KCl, and 100 mM MgCl₂ (pH=7.5).

• Store at room temperature for up to one year.

Tris Buffered Saline (TBS, 1X)

• 20 mM Tris base and 150 mM NaCl.

• Adjust the pH to 7.6 using HCl and/or NaOH.

• Store at 4 °C up to 1 year

COMMENTARY:

Background Information:

Protein phosphorylation is an important post-translational modification that plays a major role in cellular biology by influencing protein activity. To better understand protein phosphorylation and the roles that phosphoproteins play in cellular events, a key challenge is to monitor and identify the kinases that phosphorylate cellular proteins. One method available for kinase-substrate identification utilizes databases that predict substrates based on the fact that kinases recognize the primary sequence of amino acid residues surrounding a phosphosite, also called a substrate motif or consensus site, to initiate phosphorylation (Miller and Turk, 2016; Miller et al., 2008). Databases, such as NetPhorest (Miller et al., 2008), Scansite (Obenauer et al., 2003), KinasePhos (Ma et al., 2021), predict kinase-substrate pairs by comparing known substrate motif logos of the kinase to the primary sequence of the phosphoprotein. Although substrate motif predictions are fast and easy to use, without the need for experiments, a challenge is that substrate motif logos are only well characterized for a small subset of the 535 known kinases (Watson et al., 2020). As a result, the prediction of kinase-substrate pairs by substrate motif databases is biased towards characterized kinases. A second challenge is that known substrate motifs were often determined using arrays of short peptide substrates in vitro, which might not faithfully represent the preferences of kinases toward full length substrate proteins under cellular conditions (Fujii et al., 2004; Miller and Turk, 2016). Due to the lack of available knowledge, using substrate motif databases is helpful, but limited.

One method to identify kinase-substrate pairs experimentally using full length protein substrates in a cellular context involves allele-sensitive kinases from the Shokat lab. In this chemical approach, an engineered kinase uses a base-modified ATPγS analog to selectively thiophosphorylate direct substrates in a lysate mixture (Allen et al., 2007). Upon chemical alkylation of the thiophosphoryl group on the substrate protein, an epitope for a thiophosphate ester-specific antibody is generated, which is then used for immunoprecipitation of the substrates. The allele-sensitive kinase method requires engineered kinases that tolerate the bioorthogonal ATPγS analog. To date, among the nearly 535 known human kinases (Watson et al., 2020), approximately 40 mammalian kinases have been engineered to accept the ATP analog (Allen, 2008; Lopez et al., 2014), which limits the use of this method for general substrate identification.

The K-CLIP method represents a helpful alternative for kinase-substrate identification. K-CLIP is compatible with full length proteins in a variety of complex cellular mixtures. In addition, K-CLIP avoids the need for kinase mutagenesis by using a γ-phosphoryl modified ATP analog that are accepted by native kinases. Due to the reliance on native kinases, K-CLIP can be applied to a variety of different kinases and biological systems. One limitation of the K-CLIP method is the need for a good quality antibody against the kinase or substrate of interest for the immunoprecipitation step, although alternatives exist to overcome this limitation, including use of a tagged kinase or substrate (see Critical Parameters below). The availability of various complementary methods to identify kinase-substrate pairs will ultimately help improve current knowledge of the roles kinases and their phosphoprotein substrates play in cellular events.

Critical Parameters:

Selection of lysate mixture –

The choice of lysate mixture is an important factor for application of Protocols 1 and 2 to a new target phosphoprotein substrate in a complex mixture. Three key variables must be considered when choosing the lysate mixture and/or the target phosphoprotein substrate: 1) substrate abundance, 2) active phosphorylation, and 3) biological context. When considering abundance, the lysate mixture must contain the substrate of interest in sufficient quantity to visualize crosslinking by gel methods. Using lysates with sufficient endogenous levels of substrate is ideal by mimicking normal physiological conditions. In cases where the endogenous abundance of the substrates is low, the lysate mixture can be modified to obtain sufficient quantities of substrate for crosslinking. Previously in work involving p53 as the substrate of interest, the mammalian cell lysate did not contain sufficient p53, and stabilization of p53 was achieved by inhibiting the binding of MDM2, which mediates degradation (Garre et al., 2018). We also have had success using a variety of modified lysate mixtures, including lysates with the substrate of interest overexpressed with a tag or homogenates with the tagged substrate of interest exogenously added. In addition to having sufficient substrate levels, kinase(s) in the lysate must be actively phosphorylating the substrate of interest to form crosslinked substrate-kinase complexes. Prior to applying Protocols 1 and 2 to a target substrate, phosphopeptide-specific antibodies or Phos-tag SDS-PAGE (Fuji Film, Inc.) can be used to establish kinase activity and phosphorylation states of the target substrate in the lysate mixture (Sugiyama et al., 2015). The last variable to consider is the biological context. Selecting an appropriate complex lysate mixture that well represents the natural environment of the target substrate protein will help ensure that the isolated crosslinked kinase-substrate pairs are relevant to the biological system. For example, if studying a substrate target related to cancer, a lysate from a cancer-derived cell line should be used. Overall, taking time to consider these three key factors for the selection of the lysate is a critical step before performing crosslinking reactions.

Selection of enrichment methods –

In order for K-CLIP (Protocol 2) to be successful, a method to enrich the substrate of interest must be available. If the substrate of interest has a good quality antibody appropriate for immunoprecipitation, then crosslinked complexes can be isolated via direct immunoprecipitation. However, if a good quality antibody compatible with immunoprecipitation is unavailable for the substrate of interest, then an alternative enrichment method should be considered. One such method would be to express a tagged version of the substrate of interest and use an antibody specific for the tag. For example, a fusion protein tagged with FLAG can be expressed and subsequently enriched using FLAG conjugated-agarose beads (Gerace and Moazed, 2015). As another solution, particularly when expression of a tagged substrate is not possible, recombinantly-expressed and tagged substrate can be exogenously added to a lysate mixture for subsequent enrichment. For example, a hexa-histidine-tagged recombinant substrate can be added to a tissue homogenate for the crosslinking reaction, followed by enrichment using ion metal affinity chromatography (Spriestersbach et al., 2015). Overall, K-CLIP is compatible with a variety of enrichment methods, which can be designed for the most appropriate lysate or complex mixture for the substrate of interest.

Storage conditions and handling –

The quality of ATP-arylazide is crucial for successful kinase-catalyzed crosslinking experiments. Triphosphate containing compounds, such as ATP and ATP-arylazide, are prone to hydrolysis and degradation. In the case of ATP-arylazide, degradation will generate unmodified ATP or ADP, along with the arylazide linker byproduct. The ATP and ADP degradation impurities can compete with ATP-arylazide in the kinase reactions, resulting in low levels of crosslinking and poor results. More critically, the arylazide linker byproduct can participate in kinase-independent labeling (Arora and Boon, 2013), which could compromise the method by generating false positive hits. To minimize hydrolysis of ATP-arylazide, the analog should be stored as a solid at −80 °C for long-term storage (up to 12 months). Once dissolved in aqueous buffer, the analog should be stored at −80°C as small and single use aliquots to avoid freeze thaw cycles. In addition, ATP-arylazide should be kept as cold as possible while thawing and be used immediately after thawing in the kinase reaction to minimize degradation. All sample preparation and transfers should be performed on ice or in a cold room with prechilled centrifuge tubes. Even with storage at −80 °C, ATP-arylazide stored in aqueous buffer will degrade overtime and should be used within 6 months.

Troubleshooting:

Potential problems and solutions are described in Table 5.

Table 5.

Troubleshooting Possible Problems

Protocol	Problem	Possible Cause		Solution
1,2	No high molecular weight crosslinking bands or smearing observed in the experimental lane compared to the negative control lanes.	Low abundance of substrate of interest		Use more lysate or substrate of interest; if using endogenous substrate, consider use of overexpressed-tagged substrate
		Good abundance of substrate of interest	Poor antibody sensitivity	Optimize the Western blot protocol by testing different primary or secondary antibody dilutions or using a different primary antibody
			Low lysate concentration in the crosslinking reaction	Use a more concentrated lysate in the crosslinking reaction to increase the likelihood for kinase-substrate interactions.
			Low ATP-arylazide concentration in crosslinking reaction	Increase the final concentration of ATP-arylazide in the kinase reactions
			ATP-arylazide degradation, especially if older than 6 months	Use a freshly dissolved stock of ATP-arylazide
			expired kinase buffer	Make fresh 10X kinase buffer
1,2	High background in the negative control lanes, which could mask crosslinked bands in the crosslinking samples	The substrate of interest is highly abundant in the lysate		Decrease the amount of lysate used for the kinase-catalyzed crosslinking experiments
		Too much HRP developer used		Dilute the HRP developer before adding to membrane
				Use less sensitive fluorescence detection
		Too long of an exposure time on the imager		Shorten the length of exposure
		The blocking step was unsuccessful		Optimize the blocking step by trying alternative blocking reagents and incubation times
2	Low levels or undetectable levels of the substrate of interest by Western blot after immunoprecipitation	Immunoprecipitation step was unsuccessful		Use a larger quantity of primary antibody
				Use a different immunoprecipitation-compatible primary antibody
				Express the substrate with an immunoprecipitation-compatible tag
				Use a different enrichment method
2	Unequal substrate protein levels visualized by Western blot after immunoprecipitation	Accidently removed beads when performing washing steps		During the washing steps, remove small amounts of buffer sequentially to avoid removing beads
2	Unable to distinguish substrate of interest from the antibody bands on the Western blot after immunoprecipitation	The substrate of interest and the heavy chain (50 kDa) or light chain (25 kDa) of the antibody have similar molecular weights		Use either a light chain-specific or heavy-chain specific secondary antibody to visualize the Western blot

Anticipated Results:

Basic Protocol 1 –

The expected result after kinase-catalyzed crosslinking is to see both the substrate of interest and high molecular weight crosslinked bands in the membrane after visualization of the substrate by Western blot analysis. Bands for the substrate of interest should be at equal levels in all samples, which assures equal protein loading of the reactions into each lane. However, if needed, a separate gel can be used to assess a different loading control, such as GAPDH, tubulin, or actin. Higher molecular weight bands or smears indicate that the kinase-catalyzed crosslinking was successful at generating complexes between the substrate of interest and kinases or associated proteins. In total, the uncrosslinked target substrate in all samples, along with higher molecular weight crosslinked complexes in the experimental lane, confirms successful crosslinking.

As an illustrative example and useful control to practice the method, p53 crosslinking is shown here. In the p53 example, the kinase-catalyzed crosslinking experiment was performed with HEK293 lysate, which contains endogenous p53. HEK293 lysates were incubated with or without ATP-arylazide in the presence or absence of UV irradiation. Proteins in the lysate samples were separated by SDS-PAGE, followed by Western blotting to identify uncrosslinked p53 and p53-crosslinked complexes. As a loading control, equal levels of uncrosslinked p53 were observed in all lanes (Figure 3, lanes 1 – 4). High molecular weight crosslinked bands containing p53 were observed in the presence of ATP-arylazide with UV (Figure 3, lane 4). In contrast, no crosslinked complexes were visualized in the absence of UV light (Figure 3, lane 3) or ATP-arylazide (Figure 3, lane 2), which indicates that the crosslinking is dependent on the ATP analog and UV irradiation. In kinase-catalyzed crosslinking gels, the appearance of smeared instead of distinct bands for the crosslinked complexes is common.

Basic Protocol 2 –

After performing K-CLIP with gel analysis, the expected result is to see both the uncrosslinked substrate of interest and the crosslinked complexes containing the substrate by Western blot analysis after immunoprecipitation. As a load control, the substrate of interest should have equal levels in all lanes containing lysate to show that the immunoprecipitation step was successful. In the crosslinked sample with ATP-arylazide in the presence of UV, high molecular weight crosslinked complexes containing the substrate of interest should be observed.

Again, as an illustrative example, K-CLIP with endogenous p53 in HEK293 lysates is shown here. For the p53 K-CLIP example, HEK293 lysates were incubated with or without ATP-arylazide in the absence or presence of UV irradiation, followed by p53 immunoprecipitation. Enriched proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane to visualize p53 and p53-crosslinked complexes by Western blotting. The levels of p53 were equal in the samples (Figure 4, lanes 1 – 3), which indicates successful immunoprecipitation. High molecular weight crosslinked complexes specific to p53 were observed in the presence of ATP-arylazide with UV irradiation (Figure 4, lane 3). In contrast, crosslinked complexes were absent with the omission of UV light (Figure 4, lane 2) or ATP-arylazide (Figure 4, lane 1). Because p53 (53 kDa) migrates similarly to the heavy chain of the IgG antibody (50 kDa), included in the analysis was an agarose bead antibody control to distinguish p53 from the IgG bands of the antibody used for immunoprecipitation (Figure 4, lane 4). Also, as shown in the p53 example, high molecular weight crosslinked smearing is common with photocrosslinking.

Time Considerations:

Basic Protocol 1: Kinase-catalyzed crosslinking and gel analysis can be completed in roughly two days. Day one requires roughly 6 hours for kinase reaction, gel separation, electrotransfer, and incubation with primary antibody overnight, while day two requires 2 hours for Western blotting and gel visualization. Basic Protocol 2: K-CLIP with gel analysis requires 3 days. Day one will require 3 hours for kinase reaction and overnight incubation for immunoprecipitation, day 2 will require 5 hours for eluting proteins from the beads, gel separation, electrotransfer, and incubation with primary antibody overnight, and day 3 will take 2 hours for Western blotting and gel visualization.

DATA AVAILABILITY STATEMENT:

Data available on request from the authors