The binding of platelet derived growth factor (PDGF) to the PDGF receptor stimulates phosphorylation of 8 tyrosines in the receptor's cytoplasmic domain. The enzyme phosphatidylinositol 3'kinase (PI 3-kinase) binds to one or more of the phophotyrosines through its SH2 domains and is thereby activated. To identify the activation phosphotyrosines, you synthesize 8 pentapetides that contain the critical tyrosines (at the N-terninus) in their phosphorylated or unphosphorylated forms. You then mix an excess of each of the various pentapeptides with phosphorylated PDGF receptor and PI 3-kinase. Immunoprecipitation of the PDGF receptor will bring down any bound PI 3-kinase, which can be assayed by its ability to add 32P-phosphate to its substrate.
A) Draw a flow diagram showing how this assay works.
B) Do your results support the notion that PI-kinase binds to phosphotyrosines in the activated PDGF receptor? Why or why not?
C) THe amino acid sequences of the PDGF receptor pentapeptide tested above (number accodingly to the position of tyrosine in the PDGF receptor) and peptide segments that are known to bind to PI 3-kinase in other activated receptors are shown below. Which amino acid residues are most important and why?
684 YSNAL YMMMR (FGF receptor)
708 YMDMS YTHMN (insulin receptor)
719 YVPML YEVML (hepatocyte growth factor receptor)
731 YADIE YMDMK (steel factor receptor)
739 YMAPY YVEMR (CSF-1 receptor)
743 YDNYE
746 YEPSA
755 YRATL
The binding of platelet derived growth factor (PDGF) to the PDGF receptor stimulates phosphorylation of 8 tyrosines in the receptor's cytoplasmic domain. The enzyme phosphatidylinositol 3'kinase (PI 3-kinase) binds to one or more of the phophotyrosines through its SH2 domains and is thereby activated. To identify the activation phosphotyrosines, you synthesize 8 pentapetides that contain the critical tyrosines (at the N-terninus) in their phosphorylated or unphosphorylated forms. You then mix an excess of each of the various pentapeptides with phosphorylated PDGF receptor and PI 3-kinase. Immunoprecipitation of the PDGF receptor will bring down any bound PI 3-kinase, which can be assayed by its ability to add 32P-phosphate to its substrate.
A) Draw a flow diagram showing how this assay works.
B) Do your results support the notion that PI-kinase binds to phosphotyrosines in the activated PDGF receptor? Why or why not?
C) THe amino acid sequences of the PDGF receptor pentapeptide tested above (number accodingly to the position of tyrosine in the PDGF receptor) and peptide segments that are known to bind to PI 3-kinase in other activated receptors are shown below. Which amino acid residues are most important and why?
684 YSNAL YMMMR (FGF receptor)
708 YMDMS YTHMN (insulin receptor)
719 YVPML YEVML (hepatocyte growth factor receptor)
731 YADIE YMDMK (steel factor receptor)
739 YMAPY YVEMR (CSF-1 receptor)
743 YDNYE
746 YEPSA
755 YRATL
