Bloodgood R A
Department of Anatomy and Cell Biology, University of Virginia School of Medicine, Charlottesville 22908.
Exp Cell Res. 1992 Feb;198(2):228-36. doi: 10.1016/0014-4827(92)90375-i.
Crosslinking of surface-exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. Previous work has shown that these membrane glycoprotein movements are dependent on a critical concentration of free calcium in the medium and are inhibited reversibly by calcium channel blockers and the protein kinase inhibitors H-7, H-8, and staurosporine. These observations suggest that the flagellum may use a signaling pathway that involves calcium-activated protein phosphorylation to initiate flagellar membrane glycoprotein movements. In order to pursue this hypothesis, we examined the calcium dependence of phosphorylation of flagellar membrane-matrix proteins using an in vitro system containing [gamma-32P]ATP or [35S]ATP gamma S. Using only endogenous enzymes and endogenous substrates found in the membrane-matrix fraction obtained by extraction of flagella with 0.05% Nonidet P-40, we observed both calcium-independent protein phosphorylation and calcium-dependent protein phosphorylation in addition to an active protein dephosphorylation activity. Addition of micromolar free calcium increased the amount of protein phosphorylation severalfold. Calcium-activated protein kinase activity was inhibited by H-7, H-8, and staurosporine, the same protein kinase inhibitors that inhibit the calcium-dependent glycoprotein redistribution in vivo. A small group of polypeptides in the 26-58 kDa range exhibited a dramatic increase in phosphorylation in the presence of 20 microM free calcium. We suggest that Chlamydomonas utilizes the intraflagellar free calcium concentration to regulate the phosphorylation of specific flagellar proteins in the membrane-matrix fraction, one or more of which may be involved in regulating the machinery responsible for flagellar membrane glycoprotein redistribution.
某些衣藻鞭毛膜糖蛋白表面暴露结构域的交联会诱导其在鞭毛膜平面内移动。先前的研究表明，这些膜糖蛋白的移动依赖于培养基中游离钙的临界浓度，并可被钙通道阻滞剂以及蛋白激酶抑制剂H - 7、H - 8和星形孢菌素可逆抑制。这些观察结果表明，鞭毛可能利用一种涉及钙激活蛋白磷酸化的信号通路来启动鞭毛膜糖蛋白的移动。为了探究这一假说，我们使用含有[γ - 32P]ATP或[35S]ATPγS的体外系统，研究了鞭毛膜 - 基质蛋白磷酸化对钙的依赖性。仅使用从用0.05% Nonidet P - 40提取鞭毛获得的膜 - 基质部分中发现的内源性酶和内源性底物，我们观察到除了活跃的蛋白去磷酸化活性外，还存在钙非依赖性蛋白磷酸化和钙依赖性蛋白磷酸化。添加微摩尔浓度的游离钙会使蛋白磷酸化量增加数倍。钙激活蛋白激酶活性被H - 7、H - 8和星形孢菌素抑制，这与在体内抑制钙依赖性糖蛋白重新分布的蛋白激酶抑制剂相同。在20微摩尔游离钙存在的情况下，一组分子量在26 - 58 kDa范围内的小多肽的磷酸化显著增加。我们认为衣藻利用鞭毛内游离钙浓度来调节膜 - 基质部分中特定鞭毛蛋白的磷酸化，其中一种或多种蛋白可能参与调节负责鞭毛膜糖蛋白重新分布的机制。
