Freeman N L, Field J
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Cell Motil Cytoskeleton. 2000 Feb;45(2):106-20. doi: 10.1002/(SICI)1097-0169(200002)45:2<106::AID-CM3>3.0.CO;2-3.
Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.
细胞形状和运动性的控制需要肌动蛋白细胞骨架的重排。一种可能调节肌动蛋白动力学的细胞骨架蛋白是CAP（环化酶相关蛋白；CAP/Srv2p；ASP-56）。CAP最初是从酵母中分离出来的，作为RAS调节cAMP信号传导所需的腺苷酸环化酶相关蛋白。此外，CAP还主要通过肌动蛋白单体结合活性来调节肌动蛋白细胞骨架。在许多真核生物中都发现了CAP同源物，包括哺乳动物，在哺乳动物中它们也结合肌动蛋白，但对其生物学功能知之甚少。因此，我们设计了实验来研究CAP1对肌动蛋白细胞骨架的调节作用。在各种类型的固定细胞中，CAP1定位于膜皱褶和肌动蛋白应力纤维。为了研究其在活细胞中的定位，我们构建了GFP-CAP1融合蛋白，发现缺乏肌动蛋白结合区域的融合蛋白与野生型蛋白的定位方式相同。我们还在瑞士3T3成纤维细胞中用亲和纯化的抗CAP1抗体进行了显微注射研究，发现这些抗体减弱了血清对应力纤维的刺激。最后，通过单克隆抗体亲和纯化步骤从血小板中纯化的CAP1，在显微注射到血清饥饿的瑞士3T3细胞中时，刺激了应力纤维样细丝的形成。综上所述，这些数据表明CAP1促进肌动蛋白细胞骨架的组装。
