AHNAK-mediated activation of phospholipase C-gamma 1 through protein kinase C

Abstract

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by the central repeated units ( CRUs) of the AHNAK protein in the presence of arachidonic acid. Here we demonstrate that four central repeated units ( 4 CRUs) of AHNAK act as a scaffolding motif networking PLC-gamma and PKC-alpha. Specifically, 4 CRUs of AHNAK bind and activate PKC-alpha, which in turn stimulates the release of arachidonic acid near where PLC-gamma1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-gamma and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-gamma activity. Stimulation of NIH3T3 cells expressing 4 CRUs of AHNAK with phorbol 12-myristate 13-acetate resulted in the increased generation of total inositol phosphates (IPT) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IPT was completely blocked in NIH3T3 cells depleted of PLC-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta isozyme resulting in the generation of a monophasic IPT within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IPT in NIH3T3 cells expressing 4 CRUs of AHNAK. The secondary activation of PLC is likely because of the scaffolding activity of AHNAK, which is consistent with the role of 4 CRUs as a molecular linker between PLC-gamma and PKC-alpha.