Zhang Y, Luo Y, Emmett K, Snell W J
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical School, Dallas 75235, USA.
Mol Biol Cell. 1996 Apr;7(4):515-27. doi: 10.1091/mbc.7.4.515.
Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation.
在衣藻的正鞭毛型和负鞭毛型配子受精过程中，其鞭毛相互黏附后的数秒内，鞭毛腺苷酸环化酶被激活数倍，细胞融合的准备工作随即启动。我们之前的研究表明，该信号通路中的早期事件，包括腺苷酸环化酶的调控，是由磷酸化和去磷酸化作用来调节的。在此，我们描述了一种可溶的、受鞭毛黏附调控的鞭毛蛋白激酶活性。在从未黏附的正鞭毛型和负鞭毛型配子中分离得到的鞭毛的体外实验中，一种48 kDa的可溶鞭毛蛋白持续被磷酸化，但在从已黏附1分钟的正鞭毛型和负鞭毛型配子中分离得到的鞭毛中则不然。尽管在从黏附的配子中分离得到的鞭毛中存在这种48 kDa的蛋白，但我们证明其蛋白激酶因鞭毛黏附而失活。免疫印迹分析和抑制剂研究表明，未黏附的配子中的48 kDa蛋白是被一种蛋白酪氨酸激酶磷酸化的。体内实验表明蛋白酪氨酸磷酸酶抑制剂原钒酸钠会抑制受精，这表明蛋白去磷酸化可能是信号转导所必需的。这种48 kDa的蛋白及其蛋白激酶可能是将鞭毛黏附分子的相互作用与配子激活相联系的新信号通路的首批元件之一。
