The Induction of Serine/Threonine Protein Phosphorylations by a PDGFR/TrkA Chimera in Stably Transfected PC12 Cells

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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. 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. Extracellular signals constitute a fundamental biological activity by which cells communicate with their environment by responding to changes in their external milieu. In higher eukaryotes, these signals are essential for the coordination of organ/organism function and are generally regulated through electrical and chemical networks that constitute the nervous and endocrine systems, respectively (1Bradshaw R.A. Dennis E.A. Handbook of Cell Signaling. 2nd ed. Elsevier Academic Press, San Diego, CA2009: 3047Google Scholar). In the latter case, with the exception of lipid soluble messengers, e.g. steroids, the mechanism of transmission is through the activation of plasma membrane-bound receptors following specific binding of the signaling entities. These ligand-receptor complexes trigger a response by activating the intracellular domain of the receptor that is then propagated and amplified via signaling cascades of varying complexity (2Kholodenko B.N. Cell-signalling dynamics in time and space.Nat. Revs. Mol. Cell. Biol. 2006; 7: 165-176Crossref PubMed Scopus (1025) Google Scholar). The ultimate targets are usually transcription factors that are activated/deactivated, leading to modulations in gene expression. However, many intracellular proteins are affected by these transmission processes (positively or negatively) and contribute to other changes in cellular activity independent of the terminal nuclear events. The principal mechanism for the perpetration of these signaling events is via protein post-translational modification, i.e. the immediate signaling responses, as opposed to the long term changes, depend on the regulation of existing proteins (3Hunter T. Signaling - 2000 and beyond.Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2261) Google Scholar, 4Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3523) Google Scholar). The extracellular ligands are commonly, although not exclusively, soluble proteins and, in large part, consist of hormones and growth factors, that are exocytosed and act on the cells of origin (autocrine), neighboring, but different, cells (paracrine) and distant cells (endocrine); the means of transport for this last group being blood R.A. growth factors and the regulation of cell growth and Scholar). The of receptors that these a of signaling these is the of tyrosine However, are a of protein kinases with for modifications in cells T. kinase PubMed Scopus Google and many of these are downstream by the amplified signaling the amount of the total protein phosphorylation events that from external on and as in the observed distribution of on cellular proteins T. gene of the PubMed Scopus Google Scholar). 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