The induction of serine/threonine protein phosphorylations by a PDGFR/TrkA chimera in stably transfected PC12 cells.
Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.
Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94158, USA.
Jordane
J
Biarc
Robert J
RJ
Chalkley
A L
AL
Burlingame
Ralph A
RA
Bradshaw
D020816:Amino Acid Motifs; D000818:Animals; D000077214:Becaplermin; D003001:Cloning, Molecular; D006801:Humans; D007553:Isotope Labeling; D020932:Nerve Growth Factor / Q000502:physiology; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis / Q000502:physiology; D051381:Rats; D020917:Receptor, trkA / Q000737:chemistry / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*; D015854:Up-Regulation
D010750:Phosphoproteins; D020543:Proteome; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins; D000077214:Becaplermin; D013912:Threonine; D012694:Serine; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D017479:Receptors, Platelet-Derived Growth Factor