Identification of c-Src tyrosine kinase substrates in platelet-derived growth factor receptor signaling

Abstract

c-Src non-receptor tyrosine kinase is an important component of the platelet-derived growth factor (PDGF) receptor signaling pathway. c-Src has been shown to mediate the mitogenic response to PDGF in fibroblasts. However, the exact components of PDGF receptor signaling pathway mediated by c-Src remain unclear. Here, we used stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to identify Src-family kinase substrates involved in PDGF signaling. Using SILAC, we were able to detect changes in tyrosine phosphorylation patterns of 43 potential c-Src kinase substrates in PDGF receptor signaling. This included 23 known c-Src kinase substrates, of which 16 proteins have known roles in PDGF signaling while the remaining 7 proteins have not previously been implicated in PDGF receptor signaling. Importantly, our analysis also led to identification of 20 novel Src-family kinase substrates, of which 5 proteins were previously reported as PDGF receptor signaling pathway intermediates while the remaining 15 proteins represent novel signaling intermediates in PDGF receptor signaling. In validation experiments, we demonstrated that PDGF indeed induced the phosphorylation of a subset of candidate Src-family kinase substrates - Calpain 2, Eps15 and Trim28 - in a c-Src-dependent fashion.

Figure 1

A schematic illustration of the approach to identify c‐Src kinase substrates in PDGF receptor signaling using SILAC. Mouse embryonic fibroblasts (NIH 3T3) were grown in three different stable isotope‐labeled media. One population of cells growing in 12C6‐arginine‐containing medium was left unstimulated, another population of cells growing in 13C6‐arginine‐containing medium was stimulated with 100ng/ml PDGF‐BB ligand and the third population of cells growing in 13C6–15N4‐arginine‐containing medium was pre‐treated with SU6656 for 1h and then stimulated with 100ng/ml PDGF‐BB ligand. The cells were lyzed and tyrosine‐phosphorylated proteins were enriched and analyzed as described in Materials and methods.

Figure 2

Phosphotyrosine profile of NIH/3T3 cells upon treatment with SU6656 and stimulation with PDGF‐BB. Untreated NIH/3T3 cells, PDGF treated cells and SU6656+PDGF treated NIH/3T3 cells were lysed and cell extracts were subjected to affinity purification of tyrosine‐phosphorylated proteins. Cell lysates and immunoprecipitates were then resolved by SDS‐PAGE and transferred to a nitrocellulose membrane. The membranes were probed with anti‐phosphotyrosine antibodies and re‐probed with anti‐Src or anti‐Src (pY416) antibodies as shown.

Figure 3

MS spectra of proteins showing different profiles identified in our SILAC screen. The 3 spectral peaks in each figure represent the mass shift of the same peptide. (A) A doubly charged peptide with the sequence DTADGTFLVR, from PI 3 kinase, alpha (1:3.3:2.2); (B) A doubly charged peptide with the sequence YVSELILVR from PDGF receptor, beta (1:2:1.5); (C) A doubly charged peptide with the sequence DFFQNFGNVVELR from Ras‐GAP SH3‐domain‐containing protein (1:1.5:1); (D) A doubly charged peptide with the sequence IALLEEAR from Ezrin (1:1.6:1.2); (E) A triply charged peptide with the sequence LLSPFMPFVTEELFQR from valyl‐tRNA synthetase (1:1.03:0.74); (F) A doubly charged peptide with the sequence from QGGGGAGGSVPGIER from heterogeneous nuclear ribonucleoprotein M (1:1.1:1.1). While some of the proteins (A–D) show involvement in PDGF signaling pathway as c‐Src substrates, some proteins do not show any involvement in PDGF signaling pathway (E) or do not show characteristic spectra as that of c‐Src substrates (F). The ratios shown in parentheses reflect ratio of abundance of peptides across three states (light:medium:heavy).

Figure 4

Experimental validation of a subset of novel substrates identified in this study by Western blotting. NIH/3T3 cells have been grown to 80% confluence and serum‐starved for 12h followed by either no treatment, stimulation with PDGF‐BB (100ng/mL for 5min) or PDGF stimulation after preinculation with SU6656 (2μM for 1h prior to lysis or stimulation). (A) Cell lysates were subjected to immunoprecipitation using antibodies against Eps15, Cortactin, Shp2, Fyb or cPLA2 and probed by Western blotting with anti‐phosphotyrosine antibodies. (B) Cell lysates were subjected to immunoprecipitation using anti‐phosphotyrosine antibodies and probed by Western blotting with antibodies against Trim28, Ezrin, Calpain 2, respectively. The total amount of proteins in whole cell lysates across different conditions was monitored by Western blotting with specific antibodies against these proteins.