Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS One. 2010 Oct 26;5(10):e13587. doi: 10.1371/journal.pone.0013587.
Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.
受体酪氨酸激酶 (RTKs) 在配体刺激后激活多个下游胞质酪氨酸激酶。SRC 家族激酶 (SFKs) 通过 SH2 结构域与 RTK 自身磷酸化位点的相互作用被募集到激活的 RTKs，是许多 RTK 亚家族的靶点。迄今为止，还没有对这种受体激活的 SFKs 的下游底物进行系统分析。在这里，我们利用稳定同位素标记 (SILAC) 分析进行了定量质谱分析，通过比较表达 CSF-1R 或不能与 SFKs 结合的这种 RTK 的突变形式的细胞中的含磷酸酪氨酸肽，来描绘 CSF-1R 酪氨酸激酶诱导的候选 SRC 底物。这种分析鉴定了 CSF-1R 在乳腺上皮细胞中诱导的以前未表征的酪氨酸磷酸化变化，以及一组依赖于 SRC 募集到 CSF-1R 的候选底物。这些候选物中的许多可能是直接的 SRC 靶标，因为这些蛋白质中磷酸化位点周围的氨基酸类似于已知的 SRC 激酶磷酸化基序。假定的 SRC 依赖性蛋白质包括已知的 SRC 底物以及以前未被识别的 SRC 靶标。候选底物集包括涉及多个细胞过程的蛋白质，包括细胞-细胞粘附、内吞作用和信号转导。对来自乳腺癌和肺癌患者样本的磷酸蛋白质组学数据的分析鉴定了一组 SRC 依赖性磷酸化位点，这些位点与 SRC 激活强烈相关，这代表了人类肿瘤中 RTK 下游 SRC 激活的候选标志物。总之，我们的数据揭示了 CSF-1R 激活在上皮细胞中诱导的酪氨酸磷酸化的定量、特定位点变化，并确定了许多候选的 SRC 依赖性 RTK 下游磷酸化底物。
