Sagi-Eisenberg R, Traub L M, Spiegel A M, Zick Y
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Cell Signal. 1989;1(5):519-31. doi: 10.1016/0898-6568(89)90036-3.
We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.
我们先前曾报道，存在于视网膜视杆细胞（ROS）外段的鸟苷三磷酸结合蛋白转导素（TD）的纯化的与GDP结合的α亚基，可作为蛋白激酶C（PKC）的高亲和力底物（Km = 1 microM）[齐克等人（1986年）《美国国家科学院院刊》83，9294 - 9297]。在本研究中，我们证明当TD-α以其天然形式存在于完整的ROS膜中时，它会被PKC磷酸化。这种磷酸化被激活TD的GTP-γ-S抑制，这表明只有TD-α的无活性构象作为PKC的底物。实际上，钒酸盐和AlF4都能使TD-α-GDP呈现活性构象，它们抑制PKC介导的纯化的TD-α-GDP的磷酸化。我们证明纯化的TD的β亚基也作为PKC的体外底物。此外，在它们被磷酸化之后，TD-α和β都与PKC形成高亲和力复合物。当TD的α和β亚基分别被其各自的抗体免疫沉淀时，PKC与它们共沉淀，这一发现证明了这一点。PKC使另外36、48和92 kDa的ROS蛋白磷酸化，初步鉴定为视紫红质、抑制蛋白和cGMP磷酸二酯酶。综合我们的结果强烈表明，TD的磷酸化具有生理相关性，并且通过内源性ROS蛋白的磷酸化，PKC可能在调节光转导中起关键作用。
