Abstract
It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. In the current experiments, phosphatidylinositol 3,4, 5-trisphosphate (PtdIns-3,4,5-P3) was found to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K resulted in an approximately 40% increase in both intracellular IP3 generation and intracellular calcium release as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibited the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3 production and intracellular calcium release, potentially via enhanced PtdIns-4, 5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCgamma to the lipid bilayer.
Highlights
It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions
To determine whether phospholipase C␥ might interact with the lipid products of PI3K in a manner capable of modifying ligand-dependent IP3 generation and calcium signaling, we examined PtdIns-3,4,5-P3 binding to the SH2 domains of PLC␥ and found that PtdIns-3,4,5-P3 associates with high affinity with the C-terminal SH2 (CT-SH2) domain of PLC␥ and is capable of displacing the CT-SH2 domain of PLC␥ from the activated platelet-derived growth factor receptor (PDGFR)
PtdIns-3,4,5-P3 Can Bind to the CT-SH2 Domain of PLC␥ and Mediate Dissociation from the PDGFR—To determine whether PtdIns-3,4,5-P3 might interact with PLC␥ in vivo, binding of the SH2 domains of PLC␥ to PtdIns-3,4,5-P3 was examined in vitro
Summary
It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. Bae et al [11] have found that the addition of PtdIns-3,4,5-P3 can enhance phospholipase C␥-mediated PtdIns-4,5-P2 hydrolysis in vitro and that overexpression of a constitutively active form of the p110 catalytic subunit of PI3K increases intracellular IP3 levels, raising the possibility that PtdIns-3,4,5-P3 may regulate calcium signaling as well This possibility is supported by the observation that wortmannin, an inhibitor of the catalytic site of PI3K, as well as several related enzymes, diminishes the intracellular calcium transient seen in adrenal glomerulosa cells, neutrophils, and rat leukemia cells following stimulation [12,13,14,15]. Mediated calcium release by the wild-type PDGFR, but has no effect on calcium release initiated by the Y1021 PDGFR
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