Phosphospecific antibodies reveal temporal regulation of platelet-derived growth factor beta receptor signaling.
The platelet-derived growth factor beta receptor (betaPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, betaPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing betaPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.
Bernard
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Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, Massachusetts 02114, USA.
Kazlauskas
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000918:Antibody Specificity; D020134:Catalytic Domain / Q000502:physiology; D002478:Cells, Cultured; D020690:GTPase-Activating Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D018631:ras Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D020797:Receptor, Platelet-Derived Growth Factor beta; D018631:ras Proteins