title: Threonine phosphorylation of the beta 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding.
abstract: The mechanism of outside-in signaling by integrins parallels that for growth factor receptors. In both pathways, phosphorylation of a cytoplasmic segment on tyrosine generates a docking site for proteins containing Src homology 2 (SH2) and phosphotyrosine binding domains. We recently observed that phosphorylation of a threonine (Thr-753), six amino acids proximal to tyrosine 759 in beta(3) of the platelet specific integrin alpha(IIb)beta(3), inhibits outside-in signaling through this receptor. We hypothesized that the presence of phosphothreonine 753 either renders beta(3) a poor substrate for tyrosine kinases or inhibits the docking capabilities of the tyrosyl-phosphorylated form of beta(3.) The first alternative was tested by comparing the phosphorylation of beta(3) model peptides by the tyrosine kinase pp60(c-src) and we found that the presence of a phosphate group on a residue corresponding to Thr-753 did not detectably alter the kinetics of tyrosine phosphorylation. However, the presence of phosphate on this threonine inhibited the binding of Shc to tyrosyl-phosphorylated beta(3) peptide. The inhibitory effect of the phosphate group could be mimicked by substituting an aspartic acid for Thr-753, suggesting that a negative charge at this position modulates the binding of Shc and possibly other phosphotyrosine binding domain- and SH2-containing proteins. A survey of several protein kinases revealed that Thr-753 was avidly phosphorylated by PDK1 and Akt/PKB in vitro. These observations suggest that activation of PDK1 and/or Akt/PKB in platelets may modulate the binding activity and/or specificity of beta(3) for signaling molecules.
authors.affiliation: Department of Cell Biology & Anatomy, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: R I
authors.initials: RI
authors.lastname: Kirk
authors.forename: M R
authors.initials: MR
authors.lastname: Sanderson
authors.forename: K M
authors.initials: KM
authors.lastname: Lerea
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D039661:Integrin beta3; D019751:Intramolecular Transferases / Q000378:metabolism*; D007700:Kinetics; D008387:Marine Toxins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*; D013997:Time Factors; D018909:src Homology Domains
chemical_list: D015703:Antigens, CD; D039661:Integrin beta3; D008387:Marine Toxins; D010080:Oxazoles; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D013912:Threonine; C059041:calyculin A; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase