Sheffler et al. 10.1073/pnas.0600585103. |
Supporting Text
Supporting Table 2
Supporting Table 3
Supporting Figure 5
Supporting Data Set 1
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Table 4
Supporting Figure 10
Fig. 5. RSK2 knockout augments b 1-adrenergic signaling. Shown is the isoproterenol-stimulated dose response of cAMP production for RSK2-/- and RSK2+/+ fibroblasts. Data are presented as the percent of the forskolin response (defined as 100% response) in the RSK2-/- and RSK2+/+ fibroblasts.
Fig. 6. RSK2+/+ and RSK2-/- fibroblasts have few differences in global gene expression. Shown are the full microarray data of expressed gene probes plotted as RSK+/+ Fibroblast mean signal vs. RSK2-/- fibroblast mean signal. The microarray data demonstrated the expression of 13,320 gene probes in both RSK2-/- and RSK2+/+ fibroblasts. A linear regression of the data set is shown in blue as are theoretical lines showing the cutoff for a > 2-fold change in gene expression (red) and for a > 10-fold change in gene expression (gray).
Fig. 7. RSK2+/+ and RSK2-/- fibroblasts display a number of differences in cell cycle control gene expression profiles. Shown are the microarray data of cell cycle control pathways overlaid with gene-expression color criterion and fold changes from the programs GENMAPP and MAPPFINDER (Gladstone Institute, University of California, San Francisco). Gray-colored genes are those expressed in RSK2-/- and RSK2+/+ fibroblasts that show no change in gene expression. White-colored genes are not expressed in either RSK2-/- or RSK2+/+ fibroblasts. Green-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold increase in expression over the RSK2+/+ fibroblasts. Red-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold decrease in expression compared with the RSK2+/+ fibroblasts.
Fig. 8. RSK2+/+ and RSK2-/- fibroblasts do not show differences in Ga q-coupled G protein-coupled receptor (GPCR) signaling pathway gene expression. Shown are the microarray data of GPCR signaling pathways overlaid with gene-expression color criterion and fold changes from the programs GENMAPP and MAPPFINDER. Gray-colored genes are those expressed in RSK2-/- and RSK2+/+ fibroblasts that show no change in gene expression. White-colored genes are not expressed in either RSK2-/- or RSK2+/+ fibroblasts. Green-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold increase in expression over the RSK2+/+ fibroblasts. Red-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold decrease in expression compared with the RSK2+/+ fibroblasts.
Fig. 9. RSK2 knockout induces a potentiation of both basal and agonist-stimulated ERK1/2 phosphorylation. (A) Shown are representative immunoblots from a single experiment repeated four times with quivalent results. RSK2+/+ fibroblasts and RSK2-/- fibroblasts were exposed to 100 nM MDL100,907 (M) for 10 minutes, to vehicle (0 min lane) or to 10 μM 5-HT(5, 10, 15, and 30 minute lanes). The top immunoblot shows phosphorylated ERK1/2 levels with a short exposure time so that the varying time points are not overexposed. The second blot from the top shows a longer exposure of the phosphorylated ERK1/2 immunoblot to allow emphasis on the MDL100,907 (M) and vehicle treated (0) lanes. The middle immunoblot shows total ERK1/2 levels. The bottom immunoblot shows that RSK2 is absent in the RSK2-/- fibroblasts. (B) Quantification of the net pixel intensities of phosphorylated ERK1/2 normalized to total ERK1/2, expressed as the fold over RSK2+/+ fibroblast basal for the entire time course, i.e., RSK+/+ at time zero = 1.0. (C) Quantification of the net pixel intensities of osphorylated ERK1/2 normalized to total ERK1/2, expressed as the fold over RSK2+/+ fibroblast basal comparing the 0 minute (0) and 100 nM MDL100,907 (M) treated time points of RSK2+/+ fibroblasts and RSK2-/- fibroblasts. * = Statistically significant P<0.05; NS = Not statistically significant.
Fig. 10. RSK2+/+ and RSK2-/- fibroblasts do not show differences in mitogen-activated protein kinase (MAPK) cascade gene expression. Shown are the microarray data of MAPK signaling cascades overlaid with gene-expression color criterion and fold changes from the programs GENMAPP and MAPPFINDER. Gray-colored genes are those expressed in RSK2-/- and RSK2+/+ fibroblasts that show no change in gene expression. White-colored genes are not expressed in either RSK2-/- or RSK2+/+ fibroblasts. Green-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold increase in expression over the RSK2+/+ fibroblasts. Red-colored genes are those genes in the RSK2-/- fibroblasts that show a > 2-fold decrease in expression compared with the RSK2+/+ fibroblasts.
Table 2. Yeast two-hybrid analysis reveals potential 5-HT2A receptor-interacting proteins
Clone | Identity | Gene |
2.4 | Amyloid-b precursor protein intracellular domain associated protein-1a | AIDA-1A |
5.2 | Eukaryotic translation initiation factor 3, subunit 5 e | EIF3S5 |
6.1 | Neurotrophic tyrosine kinase, receptor, type 3 isoform c precursor | NTRK3 |
7.3 | Melanoma-associated antigen | MAAT1 |
21.1 | Paraoxonase 2 | PON2 |
29.1 | Microtubule associated protein 1A | MAP-1A |
33.5 | Ribosomal protein S6 kinase 2 | RSK2 |
39.4 | Nucleoside-diphosphate kinase 3 | NME3 |
43.5 | NADH dehydrogenase (ubiquinone) 1 b subcomplex | NDUFB10 |
47.5 | Protein phosphatase 5, catalytic subunit | PPP5C |
47.6 | Glutamine synthetase | GLUL |
A yeast two-hybrid screen of a human brain cDNA using the third intracellular loop (i3) of the 5-HT2A receptor as "bait" yielded a large number of putative 5-HT2A receptor-interacting proteins . These proteins were identified based on growth on triple dropout (TDO) and quadruple dropout (QDO) media (see Supporting Text for details). Plasmids were rescued and sequenced from colonies showing the most vigorous growth on QDO and the highest b-galactosidase expression. After elimination of duplicates, clones containing only untranslated regions of DNA and clones expressing proteins or fragments not restricted to expression in neuronal tissues or cells, a list of potential 5-HT2A receptor interacting proteins was generated. Shown are the original clone numbers, the identity of the clone, and the gene symbols for various potential 5-HT2A receptor-interacting proteins identified through a yeast two-hybrid screen.
Table 3. Microarray analysis of mouse RSK2+/+ and RSK2-/- fibroblasts reveals the expression of selected GPCRs
Gene name | Gene symbol |
Coagulation factor II (thrombin) receptor | F2r |
Coagulation factor II (thrombin) receptor-like 2 | F2rl2 |
b 1 adrenergic receptor | Adrb1 |
5-hydroxytryptamine (serotonin) receptor 1A | Htr1a |
Bradykinin receptor, b | Bdkrb |
Arginine vasopressin receptor 1A | Avpr1a |
Angiotensin II receptor, type 2 | Agtr2 |
Chemokine (C-X-C) receptor 3 | Cmkar3 |
Endothelial differentiation, sphingolipid G-protein-coupled receptor, 3 | Edg3 |
Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2 | Edg2 |
Endothelial differentiation sphingolipid G-protein-coupled receptor 1 | Edg1 |
Neuropeptide Y receptor Y1 | Npy1r |
Neuropeptide Y receptor Y5 | Npy5r |
G protein-coupled receptor 85 | Gpr85 |
G protein-coupled receptor 56 | Gpr56 |
G-protein coupled receptor 88 | Gpr88 |
G protein-coupled receptor 73 | Gpr73 |
G protein-coupled receptor 19 | Gpr19 |
Prostaglandin F receptor | Ptgfr |
Prostaglandin E receptor 1 (subtype EP1) | Ptger1 |
Prostaglandin I receptor (IP) | Ptgir |
Prostaglandin E receptor 4 (subtype EP4) | Ptger4 |
Melanocortin 2 receptor | Mc2r |
Melanocortin 3 receptor | Mc3r |
Gastrin releasing peptide receptor | Grpr |
Endothelin receptor type B | Ednrb |
Shown are the gene name and gene symbols for various GPCRs identified by microarray analysis of mouse RSK2+/+ and RSK2-/- fibroblast mRNA as being present in both cell lines (see Supporting Text for details)
Table 4. Microarray analysis of mouse RSK2+/+ and RSK2-/- fibroblasts reveals that phosphatase expression patterns are similar
Gene | Expression in | Gene | Expression in | Gene | Expression in |
Acp1 | = | Ppm1b1 | = | Ppp5c | = |
Acp2 | = | Ppm1b2 | = | Pppap2b | = |
Akp2 | = | Ppm1d | = | Pps | = |
CaM-Prp | = | Ppm1g | = | Ptp4a1 | = |
dis2m2 | = | Ppp1c | = | Ptp4a2 | = |
Dusp10 | = | Ppp1ca | = | Ptp4a3 | = |
Dusp12 | = | Ppp1cb | = | Ptpn1 | = |
Dusp14 | = | Ppp1cc | = | Ptpn12 | = |
Dusp6 | UP | Ppp1r11 | = | Ptpn13 | = |
Esp | UP | Ppp1r14b | = | Ptpn14 | = |
Impa1 | = | Ppp1r2 | = | Ptpn16 | = |
Impa2 | = | Ppp1r3c | = | Ptpn2 | = |
Inpp1 | = | Ppp1r7 | = | Ptpn21 | = |
Inpp5b | = | Ppp2ca | = | Ptpn9 | = |
Inpp5e | = | Ppp2cb | = | Ptpra | = |
Inppl1 | = | Ppp2r1a | = | Ptpre | DOWN |
Minpp1 | = | Ppp2r3a | = | Ptprf | = |
Mtmr4 | = | Ppp2r4 | = | Ptprg | = |
P19-Ptp | = | Ppp2r5c | = | Ptprj | UP |
Pp2ca1b | = | Ppp3c | = | Ptprk | UP |
Ppap2a | = | Ppp3ca | = | Ptprm | = |
Ppap2c | = | Ppp3cb | = | Ptprn | = |
Ppm1a | = | Ppp3cc | = | Ptprs | = |
Ppm1b | = | Ppp4c | = | Spph1 | UP |
Shown are the gene symbols for various expressed phosphatases identified by microarray analysis of mouse RSK2+/+ and RSK2-/- fibroblast mRNA (see Supporting Text for details). Differences in the microarray data of the RSK2-/- fibroblasts from the RSK2+/+ fibroblasts are as indicated: =, equivalent expression; UP, RSK2-/- expression > 2-fold greater than +/+ fibroblasts; DOWN, RSK2-/- expression > 2-fold less than RSK2+/+ fibroblasts.
Supporting Text
Yeast Two-Hybrid Analysis. The i3 loop of the human 5-HT2A receptor was amplified by PCR from the pM05 plasmid containing the human 5-HT2A receptor coding sequence (provided by T. A. Branchek, Synaptic Pharmaceutical, Paramus, NJ) using the following oligonucleotide primers (EcoRI sites bold): Y2H-i3 loop-FWD: 5¢ -AAA GAA TTC TTT CTA ACT ATC AAG TCA CT-3¢ and Y2H-i3 loop-REV: 5¢ -AAA GAA TTC CAC CTT GCA TGC CTT TTG CT-3¢ , and Taq DNA polymerase (Boehringer-Mannheim). Following EcoRI digestion and gel purification (Gene-Clean III kit, Bio 101, Vista, CA), these PCR fragments were cloned into the EcoRI site of the pAS2-1 vector (Clontech) for use as bait, using T4 DNA ligase (New England Biolabs). Proper construction of these bait plasmids was confirmed by sequencing.
For screening, a pretransformed human brain cDNA library in Saccharomyces cerevisiae strain Y187 (Clontech) was used as the target, and the GAL4-based yeast two-hybrid procedure using yeast mating was done according to the manufacturer’s instructions (Matchmaker kit, Clontech). Positive clones in the library were screened using both nutritional markers and b-galactosidase expression and were identified by sequencing of the inserts after rescue of the plasmids from yeast into Escherichia coli. Positive controls were murine p53 and SV40 large T antigen, as provided in the Clontech kit. All controls were performed as recommended by the manufacturer to confirm that the bait alone did not activate transcription, and that all strains and plasmids had appropriate phenotypes. To confirm the results obtained, target and bait plasmids were cotransformed back into yeast strain PJ69-2A using the method of Gietz et al. .
In the initial yeast two-hybrid screen of a human brain cDNA library (Clontech), 74 colonies grew on media lacking adenine, histidine, leucine, and tryptophan [quadruple-drop-out (QDO)] medium and 111 grew on media lacking histidine, leucine, and tryptophan [triple-drop-out (TDO)] medium. Of the 111 growing on TDO, 76 (68.5%) also grew on QDO. About 50% of the colonies grown initially on QDO were also positive for b-galactosidase expression, and about 35% of the colonies grown initially on TDO were positive for b-galactosidase expression. Plasmids from yeast colonies showing the most vigorous growth on QDO and the highest b-galactosidase expression were rescued into E. coli and sequenced.
To establish the region of the i3 loop that interacts with the RSK2 target, truncated i3 loop bait constructs were prepared by inserting stop codons into four positions of the i3 loop bait plasmid via site-directed mutagenesis (Stratagene Quick-Change kit at positions C268, S282, Q296, and R310 to create serial truncations of the i3 loop).
The i3 loop bait with a deletion of amino acids 270-280 was constructed through a multi-step PCR amplification. First, the primers i3-270-280-FWD (5’- CAA GTG TCT GAA GAA CAA CT-3’) and 269-BEG-i3-REV-OVER (5¢ -GAG GGA GGA AGC TGA ATA CAC ACA AAG TAG CTT CTT TC-3¢ ) were used to amplify the proximal portion of the i3 loop bait, adding a 16-bp overhang at the 3¢ end (marked in bold). The primers 281-END-i3-FWD (5¢ - TTC AGC TTC CTC CCT CAG AG-3¢ ) and i3-270-280-REV (5¢ -TTC CCG ACT GGA AAG CGG GC-3¢ ) were used to amplify the distal portion of the i3 loop bait, including a 16-bp region of overlap with the proximal PCR product (marked in bold). The proximal and distal PCR products were gel-purified and used as a template in a second round of PCR with the primers i3-270-280-FWD and i3-270-280-REV. This PCR product was gel-purified, digested with EcoRI, and gel-purified a second time. The PCR fragment was cloned into the EcoRI site of the pAS2-1 vector, using T4 DNA ligase. Proper construction and mutagenesis of the above bait plasmids were confirmed by sequencing.
Truncated "bait" constructs and the RSK2 "target" were then cotransformed into yeast strain PJ69-2A by using the method of Gietz et al. , and interactions with RSK2 were tested by nutritional and b-galactosidase screening. To compare growth of the various truncation mutants, and therefore i3 loop-RSK2 interactions, cultures of yeast cotransformants were grown overnight in leucine and tryptophan (-L-W) dropout liquid medium. Yeast cells were counted and normalized, and dilutions were prepared of the normalized yeast cultures. One microliter of the diluted cultures was spotted on QDO and -L-W agar plates. Growth was monitored after 72 hrs.
Microarray and Pathway Analysis of RSK2-/- and RSK2+/+ Fibroblasts.
Microarray analyses of RSK2-/- and RSK2+/+ fibroblasts were performed as previously described . Briefly, nearly confluent 100-mm dishes of RSK2+/+ or RSK2-/- fibroblasts were harvested under RNase free conditions. Samples were prepared by the Gene Expression Array Core Facility at Case Western Reserve University, following methods recommended by Affymetrix. Briefly, total RNA was extracted using TRIzol (Invitrogen) followed by RNA clean up using Qiagen columns. Qiagen columns were used to clean up cDNA and/or RNA as required using the manufacturer’s protocol. This was followed by cDNA synthesis using an oligo-dT primer coupled to T7 RNA polymerase promoter. Reverse transcription of RNA was performed using Superscript II reverse transcriptase in a 20-m l reaction at 42° C for 1 hr. Second-strand synthesis was carried out immediately in presence of E. coli DNA polymerase I, RNase H, and DNA ligase. The reaction mixture was incubated for 2 hr at 16oC and a further 5 min in the presence of T4 DNA polymerase. The reaction was terminated by addition of EDTA followed by cDNA clean-up using Qiagen columns and storage overnight at -20oC. In vitro transcription was used to generate complementary RNA (cRNA) using a Bioarray High Yield ENZOkit (Affymetrix) followed by cleanup of RNA samples. Purified in vitro-transcribed cRNA was subjected to fragmentation at 94oC for 35 min using 1´ fragmentation buffer (40 mM Tris acetate, pH 8.1/100 mM KOAc/30 mM MgOAc) then placed on ice. Preconditioning for hybridization was performed in 1´ hybridization cocktail (100 mM MES/1 M Na+/20 mM EDTA/0.01% Tween 20). Herring sperm and acetylated BSA were added to a final concentration of 0.1 and 0.5 mg/ml, respectively. A 15-ml aliquot of a 20´ mixture of in vitro transcripts of bacterial genes BioB, BioC, BioD, and cre were added to the cocktail to give final concentrations of 1.5, 5, 25, and 100 pM respectively. Control oligonucleotide was added to a final concentration of 50 pM. The amount of fragmentation reaction containing 15 mg of cRNA was added to the cocktail, and the remaining volume was made up with molecular biology grade water. Preconditioning of the array chip was done in 1´ hybridization buffer for 10-15 min at 45oC with rotation (45 rpm). The preconditioning buffer was then removed from chip chamber. Sample hybridization cocktail was added to the chip and hybridized overnight (16 hr) at 45oC with rotation (45 rpm). For posthybridization washing and staining, samples were recovered from the chips and stored in their original vials, and the hybridization chamber was filled with Buffer A (nonstringent: 6´ and hybridization was overnight (16 hr) at 45oC with rotation (45 rpm) in sample hybridization cocktail. For posthybridization washing and staining, samples were recovered from the chips and stored in their original vials. The hybridization chamber was filled with Buffer A (nonstringent: 6´ SSPE, 0.01% Tween 20). Modules in the Fluidics Station 400 were primed according to Affymetrix’ protocol. Samples were loaded into the modules, and washing and staining was done in stringent buffer B (10 mM MES/0.1 N Na+/0.01% Tween 20) was also used in the protocol. The streptavidin-phycoerythrin stain mix was as follows: 50 mM MES/0.5 M Na+/0.025% Tween 20/2 mg/ml acetylated BSA/10 mg/ml SAPE).The amplification (antibody) solution mix was as follows: 50 mM MES/0.5 M Na+/0.025% Tween 20/2 mg/ml acetylated BSA/0.1 mg/ml normal goat IgG/3 mg/ml biotinylated antibody. All chips were scanned twice. For data analysis, images obtained were converted into Microsoft EXCEL format using MAS 5.0 software (Affymetrix). All chips were scaled to mean target intensity of 1500. Affymetrix present (P) and absent (A) calls were used. The Affymetrix detection algorithm uses probe-pair intensities to generate a detection P value and to assign present (P), marginal (M), or absent (A) calls. Each probe pair in a probe set is considered as having a potential vote in determining the presence or absence of a measured transcript, and this vote is defined by a value called the discrimination score (R). This R score is calculated by the Affymetrix software for each probe set and compared to a predefined threshold Tau. Tau was set at the default of 0.015 for this microarray. Probe pairs with R values lower than Tau vote for the absence of the transcript and probe pairs with R values higher than Tau vote for the presence of the transcript. The voting result is summarized as a P value, with P<0.4 given a present (P) call, P>0.6 given an absent (A) call, and 0.4>P>0.6 given a marginal (M) call. For comparisons between chips, genes with 2-fold differences in expression compared to controls were considered to be significantly differentially expressed. For pathway analysis of RSK2-/- and RSK2+/+ fibroblast gene expression patterns, GENMAPP and MAPPFINDER software packages were used (www.GenMAPP.org) .
RNA Isolation and Quantitative Real-Time PCR.
RSK2+/+ and RSK2-/- fibroblasts were harvested, and total RNA was extracted by the Roche High Pure RNA Isolation kit (Roche, Indianapolis) according to the manufacturer’s specifications and quantified. Total RNA (5 mg) was reverse-transcribed with Superscript II (Invitrogen) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using the ABI Prism 7700 Sequence Detection System Cycler (Applied Biosystems). Reactions were performed in triplicate and detected by SYBR green dye (Stratagene) using the following primers. RSK2: forward primer, 5'-GTG ACC TCA GCA CTC GGG C-3'; reverse primer, 5'-GCT CGT TGC TGA TGG ACT GC-3'; b-actin (control): forward primer, 5'-CTT TGC AGC TCC TTC GTT GC-3'; reverse primer, 5'-ACG ATG GAG GGG AAT ACA GC-3'. Data were collected during each PCR cycle and analyzed using the Sequence Detection Software (Ver. 1.6.3, Applied Biosystems). Results were expressed as the molar ratio of mRNA to mouse b-actin mRNA.
Intracellular Calcium Mobilization.
RSK2+/+ and -/- fibroblasts were plated at 30,000 cells/well into 96-well plates in DMEM supplemented with 5% dialyzed fetal calf serum. Cells were assayed for intracellular Ca2+ response to agonist 24 hr after plating. The culture medium was removed by aspiration and replaced with 30 ml of Calcium Flux Assay Kit for FlexStation Dye (Molecular Devices, Sunnyvale, CA) dissolved in assay buffer (2.5 mM probenecid/20 mM Hepes/138 mM NaCl/5.3 mM KCl/1.3 mM CaCl2/0.49 mM MgCl2/0.4 mM MgSO4/0.4 mM KH2PO4/0.34 mM Na2HPO4, pH 7.4). Plates were incubated in the dye for 1 hr at 37ºC and 5% CO2. Drugs were diluted in assay buffer as 2´ stocks and aliquoted into a 96-well plate. Fluorometric imaging was performed using a FlexStation II plate reader (Molecular Devices), which transfers 30 ml from the drug plate to the cells and takes fluorescent readings for 2 min every second. Fluorescence is excited at 485 nM and emitted at 525 nM, using a 515 nM cutoff. Drugs are added 20 seconds into the plate read to establish a steady base line. Fluorescence data is obtained as relative fluorescence units (RFU). RFU measurements were baseline-subtracted and Calcium response time-courses were plotted using PRISM 4.03 software (GraphPad, San Diego).
[32P]Orthophosphate Metabolic Labeling.
RSK2-/-, RSK2+/+, RSK2-/- FLAG-5-HT2A Stable, and RSK2+/+ FLAG-5-HT2A Stable fibroblasts were split at 400,000 cells per well into six-well plates in DMEM supplemented with 5% dialyzed fetal calf serum. The following day, cells were washed with phosphate-free DMEM and then incubated in the same medium for 1 hr. Cells were then labeled with 0.1 mCi/ml [32P]orthophosphate in phosphate-free DMEM for 4 hr and treated with 10 mM 5-HT for varying times. Cells were washed three times with Tris-buffered saline (50 mM Tris/150 mM NaCl, pH 7.4) and lysed in 32P-Lysis Buffer [1.5% (w/v) CHAPS/50 mM Hepes/150 mM NaCl/1 mM EDTA/10 mM Na4P2O7/2 mM orthovanadate/1 × EDTA-free complete protease inhibitor mixture (Roche), pH 7.5] for 10 minutes on ice. Cells were harvested by scraping, clarified by centrifugation at 13,000 ´ g for 10 minutes at 4oC, and FLAG-5-HT2A was immunoprecipitated as previously described . Samples were loaded onto 10% SDS polyacrylamide gels, electrophoresed, and gels were dried. Autoradiography was used to detect [32P]orthophosphate incorporation. Western blots were prepared to demonstrate the immunoprecipitation of FLAG-5-HT2A receptors.