The TRP Ca2+ channel assembled in a signaling complex by the PDZ domain protein INAD is phosphorylated through the interaction with protein kinase C (ePKC).
Photoreceptors which use a phospholipase C-mediated signal transduction cascade harbor a signaling complex in which the phospholipase Cbeta (PLCbeta), 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 homolog 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 deduced amino acid sequence of Calliphora ePKC comprises 685 amino acids (MW = 78 036) and displays 80.4% sequence identity with Drosophila ePKC. Immunoprecipitations with anti-ePKC antibodies led to the coprecipitation of PLCbeta, TRP, INAD and ePKC but not of rhodopsin. Phorbolester- 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 presence of extracellular Ca2+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop which regulates Ca2+ influx through the TRP channel.
Huber
A
A
Zoological Institute I, University of Karlsruhe, Germany. DC05@rz.uni-karlsruhe.de
Sander
P
P
Bähner
M
M
Paulsen
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D004175:Diptera / Q000235:genetics / Q000378:metabolism; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D005136:Eye Proteins / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D050052:TRPC Cation Channels
D015220:Calcium Channels; D029721:Drosophila Proteins; D005136:Eye Proteins; D050052:TRPC Cation Channels; C091568:inaD protein, Drosophila; C096351:transient receptor potential cation channel, subfamily C, member 1; D011493:Protein Kinase C