Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells.
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.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.
Melodie L
ML
Knowlton
Laura M
LM
Selfors
Carolyn N
CN
Wrobel
Ting-Lei
TL
Gu
Bryan A
BA
Ballif
Steven P
SP
Gygi
Roberto
R
Polakiewicz
Joan S
JS
Brugge
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D016186:Receptor, Macrophage Colony-Stimulating Factor