Yoshida Y, Toyosato A, Islam M O, Koga T, Fujita S, Imai S
Department of Pharmacology, Niigata University School of Medicine, Japan.
Mol Cell Biochem. 1999 Jan;190(1-2):157-67.
A 240-kDa protein isolated from porcine aortic smooth muscle as a substrate for cGMP-dependent protein kinase (cGMP kinase) whose phosphorylation was in a close association with stimulation of partially purified plasma membrane Ca2+ -pump ATPase by the kinase was later shown to represent splicing variants of type 1 inositol 1,4,5-trisphosphate (IP3) receptor. To further clarify the role played by this protein in the stimulation of Ca2+ -pumpATPase, it was attempted in thepresent study to specifically remove the protein by immunoprecipitation with an antibody specific to type 1 IP3 receptor. Contrary to expectation, stimulation of the ATPase by cGMP kinase was still observed after removal of the IP3 receptor. Furthermore, cGMP kinase stimulated a highly purified preparation of Ca2+ -pump ATPase deprived of IP3 receptor when the concentrations of the ATPase were low enough (10-20 nM) to make it retain a monomeric form, while it did not produce stimulation when the concentration of the enzyme was increased to 40 nM at which the enzyme is known to take an oligomeric, fully activated form insensitive to activation by calmodulin. Heat-inactivated cGMP kinase and cGMP kinase without cGMP failed to stimulate the highly purified Ca2+ -pumpATPase. In addition, type Ialpha but not type Ibeta cGMP kinase was found to stimulate the ATPase. The stimulation of Ca2+ -pump ATPase by cGMP kinase occurs without any detectable phosphorylation of the ATPase. In conclusion, cGMP kinase can stimulate the plasma membrane Ca2+ -pump ATPase when it is in a monomeric form without phosphorylating the Ca2+ -pump ATPase and that of the two cGMP kinase isozymes found in the vascular smooth muscle, only type Ialpha cGMP kinase participates in the stimulation.
从猪主动脉平滑肌中分离出一种240 kDa的蛋白质，作为环磷酸鸟苷依赖性蛋白激酶（cGMP激酶）的底物，其磷酸化与该激酶对部分纯化的质膜Ca2+ -泵ATP酶的刺激密切相关，后来发现它代表1型肌醇1,4,5 -三磷酸（IP3）受体的剪接变体。为了进一步阐明该蛋白在刺激Ca2+ -泵ATP酶中所起的作用，本研究尝试用针对1型IP3受体的特异性抗体通过免疫沉淀法特异性去除该蛋白。与预期相反，去除IP3受体后仍观察到cGMP激酶对ATP酶的刺激。此外，当ATP酶浓度足够低（10 - 20 nM）以使其保持单体形式时，cGMP激酶刺激了不含IP3受体的高度纯化的Ca2+ -泵ATP酶制剂，而当酶浓度增加到40 nM时，它没有产生刺激，已知该酶在40 nM时呈寡聚的、对钙调蛋白激活不敏感的完全激活形式。热失活的cGMP激酶和不含cGMP的cGMP激酶均未能刺激高度纯化的Ca2+ -泵ATP酶。此外，发现Iα型而非Iβ型cGMP激酶能刺激该ATP酶。cGMP激酶对Ca2+ -泵ATP酶的刺激在ATP酶没有任何可检测到的磷酸化的情况下发生。总之，cGMP激酶在其处于单体形式时可刺激质膜Ca2+ -泵ATP酶，而不使Ca2+ -泵ATP酶磷酸化，并且在血管平滑肌中发现的两种cGMP激酶同工酶中，只有Iα型cGMP激酶参与这种刺激。
