AHNAK-mediated activation of phospholipase C-γ1 through protein kinase C

We have recently shown that phospholipase C-γ (PLC-γ) 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-γ and PKC-α. Specifically, 4 CRUs of AHNAK bind and activate PKC-α, which in turn stimulates the release of arachidonic acid near where PLC-γ1 is localized. Moreover, 4 CRUs of AHNAK interacted with PLC-γ and the concerted action of 4 CRUs with arachidonic acid stimulated PLC-γ 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 (IP_T) and mobilization of the intracellular calcium. Phorbol 12-myristate 13-acetate-dependent generation of IP_T was completely blocked in NIH3T3 cells depleted of PLC-γ1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-β isozyme resulting in the generation of a monophasic IP_T within 30 s in NIH3T3 cells, led to a biphasic pattern for generation of IP_T 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-γ and PKC-α.