Liu M, Parker L L, Wadzinski B E, Shieh B H
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37232-6600, USA.
J Biol Chem. 2000 Apr 21;275(16):12194-9. doi: 10.1074/jbc.275.16.12194.
In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [(32)P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells.
在果蝇视觉信号传导通路中，瞬时受体电位（TRP）钙通道、磷脂酶Cβ（无受体电位A）以及蛋白激酶C的一种眼特异性同工型（眼PKC）通过与支架蛋白INAD相互作用，组成了一个多分子信号复合体。此前，我们发现INAD与眼PKC之间的相互作用是光反应失活的前提条件，这表明眼PKC使复合体中的蛋白质发生磷酸化。为了鉴定眼PKC的底物，我们使用抗INAD抗体从头部裂解物中免疫沉淀该复合体，并进行体外激酶分析。用[（32）P]ATP孵育野生型免疫复合体，结果显示TRP和INAD发生了磷酸化。相比之下，来自缺失眼PKC的inaC突变体的免疫复合体未显示TRP或INAD的磷酸化。我们还研究了可能参与复合体中蛋白质去磷酸化的蛋白磷酸酶。蛋白磷酸酶抑制剂冈田酸、微囊藻毒素和蛋白磷酸酶抑制剂-2可部分抑制TRP和INAD的去磷酸化。这些磷酸酶活性在野生型头部的细胞质中富集，但在缺乏光感受器的玻璃突变体提取物中则大幅降低。我们的研究结果表明，INAD作为活化PKC的受体（RACK）发挥作用，使眼PKC能够磷酸化INAD和TRP。此外，INAD和TRP的去磷酸化由优先在光感受器细胞中表达的PP1/PP2A样酶催化。
