Lee In Hye, You Je Ok, Ha Kwon Soo, Bae Duk Soo, Suh Pann-Ghill, Rhee Sue Goo, Bae Yun Soo
Division of Molecular Life Sciences, Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, Korea.
J Biol Chem. 2004 Jun 18;279(25):26645-53. doi: 10.1074/jbc.M311525200. Epub 2004 Mar 19.
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 (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-gamma1 by RNA interference. Furthermore, bradykinin, which normally stimulated the PLC-beta 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-gamma and PKC-alpha.
我们最近发现，在花生四烯酸存在的情况下，磷脂酶C-γ（PLC-γ）可被AHNAK蛋白的中央重复单元（CRU）激活。在此我们证明，AHNAK的四个中央重复单元（4个CRU）作为一个支架基序，将PLC-γ和蛋白激酶C-α（PKC-α）连接成网络。具体而言，AHNAK的4个CRU结合并激活PKC-α，进而刺激花生四烯酸在PLC-γ1定位处附近释放。此外，AHNAK的4个CRU与PLC-γ相互作用，并且4个CRU与花生四烯酸的协同作用刺激了PLC-γ的活性。用佛波醇12-肉豆蔻酸酯13-乙酸酯刺激表达AHNAK的4个CRU的NIH3T3细胞，导致总肌醇磷酸（IP(T)）生成增加以及细胞内钙的动员。在通过RNA干扰耗尽PLC-γ1的NIH3T3细胞中，佛波醇12-肉豆蔻酸酯13-乙酸酯依赖性的IP(T)生成被完全阻断。此外，缓激肽通常刺激PLC-β同工酶，在NIH3T3细胞中30秒内导致单相IP(T)的生成，但在表达AHNAK的4个CRU的NIH3T3细胞中却导致IP(T)生成呈现双相模式。PLC的二次激活可能是由于AHNAK的支架活性，这与4个CRU作为PLC-γ和PKC-α之间的分子连接物的作用一致。
