Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

Abstract

Recent studies have shown that ligand-activated growth factor receptors as well as transforming versions of nonreceptor protein-tyrosine kinases physically associate with phosphatidylinositol-3 kinase (PI-3 kinase).Reasoning that PI-3 kinase might also play a role in the normal functions of nonreceptor kinases, we sought to determine whether association with PI-3 kinase might serve as a measure of nonreceptor protein-tyrosine kinase activation under physiological conditions. We found that p60fcsrc as well as p59fy&, the product of another member of the src family of proto-oncogenes, physically associated with a PI kinase activity within 5 s after exposure to thrombin. Furthermore, PI kinase reaction products generated in p6O-SFC, p60CSrc or p590' containing immunoprecipitates were indistinguishible, demonstrating the identity of the associated enzyme as PI-3 kinase. These findings demonstrate a thrombin-dependent interaction between p60'src or p590' and PI-3 kinase and suggest a role for nonreceptor protein-tyrosine kinases in human platelet signal transduction.

Functional roles of Akt signaling in mouse skin tumorigenesis

“…The PI-3K reaction was subsequently performed as previously described (Gutkind et al, 1990). After thin layer chromatography on LK6D plates (Whatman, Ltd., Maidstone, UK), 32 P-labeled phospholipids were visualized by autoradiography and quanti®ed using a Phosphorimager (Bio-Rad).…”

Rac1 and RhoG promote cell survival by the activation of PI3K and Akt, independently of their ability to stimulate JNK and NF-κB

“…Total cellular lysates were then immunoprecipitated with anti-AU5 (Covance, Richmond, CA, USA) and an in vitro PI3-kinase reaction was performed using phosphatidyl-inositol as a substrate as previously described (Gutkind et al, 1990), except all bu ers were supplemented with 10 mM MgCl 2 to preserve interactions between G proteins and PI3K. 32 P-labeled phospholipids were separated by thin layer chromatography onto LK6D plates (Whatman, Clifton, NJ, USA), and visualized by autoradiography.…”

Phosphatidylinositol 4,5‐bisphosphate specifically stimulates PP60c‐src catalyzed phosphorylation of gelsolin and related actin‐binding proteins

“…Next to autophosphorylation [8] resulting in its activation, pp60 c-src is known to phosphorylate a number of other target proteins including ppl25 focal adhesion kinase [9], p80/85 cortactin [10,11], an actin filament-binding protein [12], the ras GTPase activating protein [13] and possibly also the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3 kinase) [14].…”