Evans Nathan J, Walker Jeffery W
Department of Physiology, University of Wisconsin--Madison, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Exp Biol Med (Maywood). 2006 Jun;231(6):882-7.
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskeletal interface. However, the dynamic properties of distinct pools of PIP2 are poorly understood, especially for PIP2 that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP2 in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP2 was analyzed using an anti-PIP2 antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP2, including profilin-1 (approximately 15 kDa), capZ (approximately 32 kDa), gCap39, (approximately 39 kDa) and alpha-actinin (approximately 106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP2 bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or alpha-actinin-bound PIP2 levels. To further explore the dynamics of PIP2 binding, brefeldin-A (BFA) was used to disrupt PIP2 binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 microM BFA increased the PIP2 signal on profilin-1 x 54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP2 bound to capZ or to alpha-actinin. Taken together, the data indicate that profilin-1 binds PIP2 dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP2 availability for Ca2+/PKC signaling in the heart.
磷脂酰肌醇-4,5-二磷酸（PIP2）是内皮素信号通路的关键下游底物，在调节膜-细胞骨架界面的蛋白质功能中发挥作用。然而，不同池的PIP2的动态特性仍知之甚少，特别是与细胞骨架蛋白结合的PIP2。我们研究了内皮素-1（ET-1）刺激对心肌中与蛋白结合的PIP2的影响。将分离的大鼠心肌细胞和匀浆的小鼠心室暴露于10 nM ET-1不同时间段，使用抗PIP2抗体和蛋白质印迹法分析与蛋白结合的PIP2。发现几种细胞骨架蛋白含有紧密结合的PIP2，包括原肌球蛋白-1（约15 kDa）、帽Z蛋白（约32 kDa）、gCap39（约39 kDa）和α-辅肌动蛋白（约106 kDa）。有趣的是，ET-1预处理15分钟后，与原肌球蛋白-1结合的PIP2量减少了46%，随后在60分钟后恢复到接近基础水平。ET-1对与帽Z蛋白、gCap39或α-辅肌动蛋白结合的PIP2水平没有影响。为了进一步探索PIP2结合的动态特性，使用布雷菲德菌素A（BFA）破坏PIP2与ADP-核糖基化因子的结合并损害受体内化。用1 μM BFA预处理15分钟后，原肌球蛋白-1上的PIP2信号增加了54%，随后在60分钟后降至基础水平以下。与ET-1一样，BFA对与帽Z蛋白或α-辅肌动蛋白结合的PIP2水平没有影响。综上所述，数据表明原肌球蛋白-1动态结合PIP2，并可能作为心脏中细胞骨架完整性和Ca2+/蛋白激酶C信号传导的PIP2可用性之间平衡的关键调节因子。
