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.

Abstract

Abstract. Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29 % of the p85o~ regulatory subunit of phosphoinositide 3-kinase (Ptdlns 3-kinase) and is accompanied by a significant increase in Ptdlns 3-kinase activity in this subcellular fraction. Actually, Ptdlns(3,4)P2 accumulation and Ptdlns 3-kinase, pp60 ~-s'~, and p125 eA~ translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of Ptdlns(3,4)P2 and the relocalization of Ptdlns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85o~ was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85a. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85et with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with Ptdlns 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 p85tx with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125 rAK. In addition, we show that Ptdlns 3-kinase is significantly activated by the p125 rAK proline-rich sequence binding to the src homology 3 domain of p85a subunit. This interaction may represent a new mechanism for Ptdlns 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 o~ro/~3 integrin and platelet aggregation evoked by thrombin.T HE accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) m and Ptdlns(3,4,5)P3 in thrombin-stimulated human platelets is now well established (16,24,38,41) and seems to be regulated differently (38). However, the exact mechanism of activation of phosphoinositide 3-kinase (Ptdlns 3-kinase) in these ceils is still obscure.

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling

“…Expression of NEP in these cells is also associated with a loss of the interaction of p85 with FAK. Numerous studies have suggested that FAK associates with PI3-K (23,26,32) and this association affects the integrity of the actin cytoskeleton (32) and cell migration (26), although it remains controversial whether PI3-K-mediated cell migration is dependent on FAK (26,33). Thus, we hypothesize that in PC cells NEP's effect on cell migration and FAK phosphorylation is also mediated by inhibition of FAK-PI3-K interaction.…”

Focal adhesion kinase in integrin-mediated signaling

“…In addition to its p110 binding site, the p85 subunit contains SH2 and SH3 domains which mediate its interactions with other cellular proteins. Association of FAK with PI 3-kinase in response to integrin activation has been demonstrated in both platelets (66) and fibroblasts (67). In addition, FAK/PI 3-kinase association in fibroblasts is stimulated by cell treatment with plateletderived growth factor (PDGF) (68), suggesting a mechanism of cross-talk between integrin and growth factor signaling pathways.…”

The Metabotropic Glutamate Receptor Activates the Lipid Kinase PI3K in Drosophila Motor Neurons Through the Calcium/Calmodulin-Dependent Protein Kinase II and the Nonreceptor Tyrosine Protein Kinase DFak

“…These phosphorylation events can activate Pyk2 by enabling subsequent tyrosine phosphorylations (particularly at Tyr402) via mechanisms that are incompletely understood (Della Rocca et al 1997;Soltoff 1998;Zwick et al 1999;Heidinger et al 2002;Fan et al 2005;Montiel et al 2007). It has also been well established that Fak and Pyk2, when activated by tyrosine phosphorylation, are each able to activate PI3K: tyrosine-phosphorylated Fak binds p85, the PI3K regulatory subunit, via both the SH3 and SH2 domains (Chen and Guan 1994;Guinebault et al 1995;Chen et al 1996). In addition, both tyrosine-phosphorylated Fak and Pyk2 are capable of activating Ras via the conserved Grb2-SoS pathway (Schlaepfer et al 1994;Dikic et al 1996;Avraham et al 2000;Rocic et al 2001), which could in principle lead to the Ras-dependent, p85-independent activation of PI3K.…”

“…CaMKII also phosphorylates mammalian Fak on C-terminal serine residues, although the functional significance of these phosphorylation events remains unknown (Fan et al 2005). Furthermore, both Pyk2 and Fak are capable of activating PI3K (Chen and Guan 1994;Schlaepfer et al 1994;Guinebault et al 1995;Chen et al 1996;Dikic et al 1996;Avraham et al 2000;Rocic et al 2001;Montiel et al 2007). Two CaMKII phosphorylation sites on Fak, serines 843 and 910, are conserved in DFak, raising the possibility that CaMKII phosphorylates DFak at these serine positions, which leads to DFak and then PI3K activation (Grabbe et al 2004).…”