Modulation of Epidermal Growth Factor stimulated ERK phosphorylation and cell motility by inositol trisphosphate kinase

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF‐receptor [EGFR] and activation of phospholipase C‐γ, resulting in the breakdown of phosphatidylinositol(4,5)‐bisphosphate and generating inositol (1,4,5)‐trisphosphate [IP 3 ] and diacylglycerol. IP 3 thus formed can be further converted to inositol (1,3,4,5)‐tetrakisphosphate [IP 4 ] by an enzyme called IP 3 ‐kinase [IP 3 K]. In this study we have investigated the effect of modulation of intracellular IP 3 K activity by the use of an inhibitor, 2‐trifluoromethyl [6‐(4‐nitrobenzyl)‐purine] [IP 3 KI] and siRNA against IP 3 KB on EGF‐induced ERK‐phosphorylation and cell motility. EC cells were stimulated with EGF in presence or absence of IP 3 KI. Cells were lysed, and proteins separated by SDS gel electrophoresis; the western blots were probed with polyclonal antibody to designated proteins. IP 3 KI, at 10 μM and 20 μM concentrations inhibited both EGF‐stimulated ERK phosphorylation and motility in a dose‐dependent manner. Interestingly, at the above concentrations, IP3KI also inhibited EGFR phosphorylation at Y1173. To further establish whether any of the above effects of IP 3 KI can be attributed to the IP 3 K activity rather than the direct inhibition of EGFR kinase by IP 3 KI, we downregulated intracellular IP 3 KB with siRNA. Lipofectamine transfection of the EC with 50 pmole and 75 pmole siRNA against IP 3 KB was accompanied by decreased level of IP 3 KB [as determined by western blot], as well as inhibition of phosphorylation of both EGFR [at Y1173] and ERK1/2. Based on these data, we conclude that IP 3 KB, and therefore its resulting product IP 4 , is capable of modulating EGF stimulated response at the receptor level.