Threonine phosphorylation of the beta 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding.
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.
Mesh Terms:
3-Phosphoinositide-Dependent Protein Kinases, Amino Acid Sequence, Antigens, CD, Binding Sites, Blood Platelets, Blotting, Western, CDC2 Protein Kinase, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Humans, Integrin beta3, Intramolecular Transferases, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxazoles, Peptides, Phosphorylation, Phosphotyrosine, Platelet Aggregation, Platelet Membrane Glycoproteins, Protein Binding, Protein Kinase C, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins pp60(c-src), Signal Transduction, Threonine, Time Factors, src Homology Domains
3-Phosphoinositide-Dependent Protein Kinases, Amino Acid Sequence, Antigens, CD, Binding Sites, Blood Platelets, Blotting, Western, CDC2 Protein Kinase, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Humans, Integrin beta3, Intramolecular Transferases, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxazoles, Peptides, Phosphorylation, Phosphotyrosine, Platelet Aggregation, Platelet Membrane Glycoproteins, Protein Binding, Protein Kinase C, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins pp60(c-src), Signal Transduction, Threonine, Time Factors, src Homology Domains
J. Biol. Chem.
Date: Oct. 06, 2000
PubMed ID: 10896934
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