Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

J Cell Biol. 1995 May;129(3):831-42. doi: 10.1083/jcb.129.3.831.

Abstract

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actin Cytoskeleton / physiology
  • Actins / metabolism
  • Avian Proteins*
  • Biological Transport
  • Blood Platelets / drug effects
  • Blood Platelets / enzymology
  • Cell Adhesion Molecules / metabolism
  • Cell Compartmentation*
  • Cytoskeletal Proteins / metabolism*
  • Cytoskeleton / physiology*
  • Enzyme Activation
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • Humans
  • Integrins / metabolism
  • Multienzyme Complexes / metabolism
  • Phosphatidylinositol 3-Kinases
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Platelet Activation / physiology*
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Protein-Tyrosine Kinases / metabolism
  • Proto-Oncogene Proteins pp60(c-src) / metabolism
  • Signal Transduction / physiology*
  • Thrombin / pharmacology
  • Tyrosine / metabolism

Substances

  • Actins
  • Avian Proteins
  • Cell Adhesion Molecules
  • Cytoskeletal Proteins
  • Integrins
  • Multienzyme Complexes
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • cytoskeletal p85 protein, chicken
  • Tyrosine
  • Phosphatidylinositol 3-Kinases
  • Phosphotransferases (Alcohol Group Acceptor)
  • Protein-Tyrosine Kinases
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • PTK2 protein, human
  • Proto-Oncogene Proteins pp60(c-src)
  • Thrombin