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. 2015 Aug 6;34(32):4260-9.
doi: 10.1038/onc.2014.361. Epub 2014 Nov 10.

Iterative Tyrosine Phosphorylation Controls Non-Canonical Domain Utilization in Crk

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Free PMC article

Iterative Tyrosine Phosphorylation Controls Non-Canonical Domain Utilization in Crk

G Sriram et al. Oncogene. .
Free PMC article

Abstract

Crk, the prototypical member of a class of Src homology-2 (SH2) and Src homology-3 (SH3) domain containing proteins that controls the coordinated assembly of signaling complexes, is regulated by phosphorylation of Y221 in the linker region, which forms an intramolecular SH2-pY221 auto-clamp to interrupt SH2-N-terminal SH3 domain (SH3N) signaling. Here, we show using LC-MS/MS and by generating phospho-specific antibodies that, iteratively with Y221, the Crk C-terminal SH3 domain (SH3C) is routinely phosphorylated on Y239 and/or Y251 by several extracellular stimuli known to engage Crk. Although phosphorylation at Y221 auto-inhibits the Crk SH2, phosphorylation of the SH3C generates an unconventional phosphoSH3C-SH3N unit in which the SH3N is fully functional to bind polyproline type II ligands and the phosphoSH3C binds de novo to other SH2 domains. Using high-throughput SH2 domain profiling, artificial neural network and position-specific scoring matrix-based bioinformatics approaches, and unbiased mass spectometry, we found that the phosphoSH3C binds several SH2 domain containing proteins, including specific non-receptor tyrosine kinases-Abl via pY251 and C-terminal Src kinase via pY239. Functionally, we show that the phosphoSH3C modulates the Abl-mediated phenotypes of cell spreading and motility. Together, these studies describe a versatile mechanism wherein phosphorylation of Crk at Y221 is not an off switch but redirects signaling from the SH2-SH3N axis to a phosphoSH3C-SH3N axis, with the SH3N as a common denominator.

Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Identification of phosphorylation sites pY221, pY239 and pY251 on Crk by LC-MS/MS
A, MS/MS spectrum of a quadruply-charged ion (m/z 1371.16) corresponding to a doubly phosphorylated peptide formula image is shown. The phosphorylation sites are Y221 and Y239. B, MS/MS spectrum of a doubly-charged ion (m/z 1055.53) corresponding to the peptide sequence formula image with a phosphorylation modification at Y251 is shown. The observed y- and b-ion series confirmed the peptide sequence and modification. C, GST-Crk was in vitro phosphorylated by immunoprecipitated Abl 1b in a kinase reaction and samples were analyzed by western blotting with anti-pY221 Crk (bottom), anti-pY239 Crk (middle) and anti-pY251 Crk (top) antibodies. D, Locations of Y239 and Y251 on the SH3C and Y221 on the inter-SH3 linker are depicted on the solution structure of Crk (PDB ID: 2EYZ).
Figure 2
Figure 2. RTKs show distinct preferences for phosphorylation of Crk at Y221/Y239/Y251
A and C, 293T cells were transfected with the plasmids indicated and lysates were analyzed by western blotting with the antibodies shown on the left. B, MDA-MB-468 cells were stimulated with EGF (panel 1), MDA-MB-231 cells with HGF (panel 2) and NIH 3T3 cells with PDGF-BB (panel 3) for the times indicated or plated on Poly-D-Lysine or Fibronectin coated dishes for 20 minutes (panel 4). Samples were analyzed by western blotting with the antibodies indicated on the left. D, Summary of Crk phosphorylation patterns observed. Red, green and blue indicate positive for pY221, pY239 and pY251 respectively.
Figure 3
Figure 3. Crk SH3N is accessible to ligands in pY221-Crk
A, 1H-15N chemical shift differences between Crk and pCrk. ∆δ values are on y-axis and amino acid numbers are on the x-axis. Red bars reflect the residues with significant line broadening or for which the perturbation value cannot be computed. B, (Left) Overlay of 1H-15N HSQC of Crk (blue) and Crk + PPII ligand (green). (Right) Overlay of 1H-15N HSQC of pCrk (red) and pCrk + PPII ligand (yellow). C, ITC profiles of Crk (top) and pCrk (bottom) with the PPII peptide ligand. Kd values are displayed as inset. D, Pull downs with purified GST, unphosphorylated GST-Crk and in vitro phosphorylated GST-Crk from 293T cell lysates expressing Flag-paxillin. Samples were analyzed with anti-DOCK180, anti-Flag and anti-pY221 Crk antibodies, anti- pY239 Crk and anti-Crk antibodies. Quantification is shown with binding of DOCK180/flag-paxillin to GST control set to zero and binding to unphosphorylated Crk set to 1. E, 293T cells were transfected with the plasmids indicated and lysates were analyzed by western blotting with the antibodies shown on the left. S-S refers to the Crk SH3N-linker SH3C construct (amino acids 123–304).
Figure 3
Figure 3. Crk SH3N is accessible to ligands in pY221-Crk
A, 1H-15N chemical shift differences between Crk and pCrk. ∆δ values are on y-axis and amino acid numbers are on the x-axis. Red bars reflect the residues with significant line broadening or for which the perturbation value cannot be computed. B, (Left) Overlay of 1H-15N HSQC of Crk (blue) and Crk + PPII ligand (green). (Right) Overlay of 1H-15N HSQC of pCrk (red) and pCrk + PPII ligand (yellow). C, ITC profiles of Crk (top) and pCrk (bottom) with the PPII peptide ligand. Kd values are displayed as inset. D, Pull downs with purified GST, unphosphorylated GST-Crk and in vitro phosphorylated GST-Crk from 293T cell lysates expressing Flag-paxillin. Samples were analyzed with anti-DOCK180, anti-Flag and anti-pY221 Crk antibodies, anti- pY239 Crk and anti-Crk antibodies. Quantification is shown with binding of DOCK180/flag-paxillin to GST control set to zero and binding to unphosphorylated Crk set to 1. E, 293T cells were transfected with the plasmids indicated and lysates were analyzed by western blotting with the antibodies shown on the left. S-S refers to the Crk SH3N-linker SH3C construct (amino acids 123–304).
Figure 4
Figure 4. SH2 domain interaction landscapes of the pY239 and pY251 motifs
A, Select hits from the rosette assay are shown for the pY239 and pY251 phosphopeptides. ‘Cont’ is a control unphosphorylated peptide. ‘Cont-P’ is a control phosphorylated peptide. N1-P is a non-specific phosphopeptide. B, Comparison of the top 10 hits of the pY239 phosphopeptide with those of the pY251 phosphopeptide. Top, Overlapping hits. Middle, pY239 specific hits. Bottom, pY251 specific hits. C, Selectivity indices of the overlapping hits (top), pY239 specific hits (middle) and pY251 specific hits (bottom). D, SH2 interaction landscapes of the pY239 (top) and pY251 motifs (bottom) obtained using NetPhorest. Y-axis indicates Probability Score. Top 10 hits are shown. E, Selectivity indices of the pY239 specific hits (top) and the pY251 specific hits (bottom). F, top, Human Crk SH3C (230–304) was phosphorylated in the TKB1 strain and used as bait for pull downs from MDA-MB-468 cell lysate. UI – Uninduced, IAA – Indoleacrylic Acid. Bottom, Phosphorylation status was analyzed by western blotting with the anti-pY251 and anti-pY239 Crk antibodies. G, Top, SYPRO RUBY stained gel of the samples indicated after pull downs with pGST, GST-SH3C or GST-pSH3C. Bottom, Western blot analysis with the anti-Csk antibody. H, Top, SYPRO Ruby stained gel of the samples obtained after pull downs with the pSH3C WT, Y239F, Y251F or Y239F/Y251F. Bottom, Western blot analysis with the anti-Csk antibody. I, 293T cells were transfected with the plasmids indicated and lysates were analyzed by western blotting with the antibodies shown on the left.
Figure 4
Figure 4. SH2 domain interaction landscapes of the pY239 and pY251 motifs
A, Select hits from the rosette assay are shown for the pY239 and pY251 phosphopeptides. ‘Cont’ is a control unphosphorylated peptide. ‘Cont-P’ is a control phosphorylated peptide. N1-P is a non-specific phosphopeptide. B, Comparison of the top 10 hits of the pY239 phosphopeptide with those of the pY251 phosphopeptide. Top, Overlapping hits. Middle, pY239 specific hits. Bottom, pY251 specific hits. C, Selectivity indices of the overlapping hits (top), pY239 specific hits (middle) and pY251 specific hits (bottom). D, SH2 interaction landscapes of the pY239 (top) and pY251 motifs (bottom) obtained using NetPhorest. Y-axis indicates Probability Score. Top 10 hits are shown. E, Selectivity indices of the pY239 specific hits (top) and the pY251 specific hits (bottom). F, top, Human Crk SH3C (230–304) was phosphorylated in the TKB1 strain and used as bait for pull downs from MDA-MB-468 cell lysate. UI – Uninduced, IAA – Indoleacrylic Acid. Bottom, Phosphorylation status was analyzed by western blotting with the anti-pY251 and anti-pY239 Crk antibodies. G, Top, SYPRO RUBY stained gel of the samples indicated after pull downs with pGST, GST-SH3C or GST-pSH3C. Bottom, Western blot analysis with the anti-Csk antibody. H, Top, SYPRO Ruby stained gel of the samples obtained after pull downs with the pSH3C WT, Y239F, Y251F or Y239F/Y251F. Bottom, Western blot analysis with the anti-Csk antibody. I, 293T cells were transfected with the plasmids indicated and lysates were analyzed by western blotting with the antibodies shown on the left.
Figure 5
Figure 5. Phosphorylation of the Crk SH3C at Y251 inhibits cell spreading on fibronectin and promotes cell motility towards EGF
A, Crk (−/−) MEFs stably reconstituted with EYFP, Crk WT or Crk Y251F were plated on fibronectin coated dishes for 20 minutes. Lysates were analyzed by western blotting with the antibodies shown. Quantification of pY245 on hyperactive Abl is shown on the right. All samples were run on the same gel and hence are comparable. However, the EYFP and WT lanes were not adjacent to the Y251F lane and hence have been demarcated by straight lines at the edges. B, MDA-MB-468 cells stably expressing EYFP, EYFP-Crk WT, EYFP-Crk Y251F were stimulated with EGF for 5 minutes and lysates were analyzed by western blotting with the antibodies shown. Quantification of pY245 Abl normalized to total Abl is shown on the right. C, Cells in B were analyzed for their motility towards EGF in a transwell Boyden chamber assay. ** indicates p<0.05. Representative images are shown on the left. D, Top, Cells in A were plated on fibronectin coated E-plates in triplicate and real-time cell spreading was recorded using xCelligence. Shown is a representative of two independent experiments each performed in triplicate. Quantification of cell index is shown as mean +/− SD. ‘**’ indicates p<0.05. Middle and Bottom, Cells in B in addition to MDA-MB-468 stably expressing Crk shRNA (Crk KD) were seeded into CIM-plates in quadruplicate in the middle panel and in duplicate in the bottom panel with an EGF gradient of 10 ng/ml and cell motility was recorded in real time using xCelligence. Middle panel is a representative of four independent experiments each performed in quadruplicate while the bottom panel is a representative of two independent experiments each performed in duplicate. Quantification of cell index is shown as mean +/− SD in the middle panel and as average of duplicates in the bottom panel. ’**’ indicates p<0.05.
Figure 6
Figure 6. Model depicting a non-canonical signaling scheme for Crk mediated by pY239 and pY251
Our data predicts that pY221 would turn off the SH2-SH3N axis while iteratively phosphorylated pY239/pY251 would turn on the pSH3C – SH3N signaling axis predicting a dynamic turnover and/or relocalization of protein complexes. In light of our findings and other independent reports, atleast two modes of auto-inhibition may exist - pY221 alone in the absence of pY239/pY251 on the SH3C or alternate auto-inhibitory domain organization that blocks SH3N accessibility (leftmost two scenarios).

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