THE TRP CA2-KINASE-C (EPKC)( CHANNEL ASSEMBLED IN A SIGNALING COMPLEXBY THE PDZ DOMAIN PROTEIN INAD IS PHOSPHORYLATED THROUGH THE INTERACTION WITH PROTEIN)

Photoreceptors which use a phospholipase C-mediated signal transductio n cascade harbor a signaling complex in which the phospholipase C beta (PLC beta), the light-activated Ca2+ channel TRP, and an eye-specific protein kinase C (ePKC) are clustered by the PDZ domain protein INAD, Here we investigated the function of ePKC by cloning the Calliphora h omolog of Drosophila ePKC, by precipitating the TRP signaling complex with anti-ePKC antibodies, and by performing phosphorylation assays in isolated signaling complexes and in intact photoreceptor cells. The d educed amino acid sequence of Calliphora ePKC comprises 685 amino acid s (MW = 78 036) and displays 80.4% sequence identity with Drosophila e PKC. Immunoprecipitations with anti-ePKC antibodies led to the copreci pitation of PLC beta, TRP, INAD and ePKC but not of rhodopsin, Phorbol ester- and Ca2+-dependent protein phosphorylation revealed that, apart from the PDZ domain protein INAD, the Ca2+ channel TRP is a substrate of ePKC. TRP becomes phosphorylated in isolated signaling complexes. TRP phosphorylation in intact photoreceptor cells requires the presenc e of extracellular Ca2+ in micromolar concentrations, It is proposed t hat ePKC-mediated phosphorylation of TRP is part of a negative feedbac k loop which regulates Ca2+ influx through the TRP channel. (C) 1998 F ederation of European Biochemical Societies.