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)

Abstract

Photoreceptors which use a phospholipase Cmediated signal transduction cascade harbor a signaling complex in which the phospholipase CL L (PLCL L), the light-activated Ca P+ 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 PLCL L, TRP, INAD and ePKC but not of rhodopsin. Phorbolester-and Ca P+ -dependent protein phosphorylation revealed that, apart from the PDZ domain protein INAD, the Ca P+ 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 Ca P+ in micromolar concentrations. It is proposed that ePKC-mediated phosphorylation of TRP is part of a negative feedback loop which regulates Ca P+ influx through the TRP channel.z 1998 Federation of European Biochemical Societies.

Phototransduction and retinal degeneration in Drosophila

“…Ca 2+ influx through the TRP channels is much higher in the inaC mutant than in the wild-type, indicating that INAC is required for inactivation of the TRP channels [161]. TRP is phosphorylated by INAC [164,165] and a mutation that abolishes the primary INAC-dependent phosphorylation site in TRP causes slow termination of the light response [166]. These data suggest that INAC contributes to deactivation, at least in part by directly phosphorylating the TRP channel.…”

“…At least three members of the signalplex are phosphorylated by PKC: NINAC, INAD and TRP [164,165]. In an in vitro assay, PKC phosphorylation of TRP is promoted by INAD and mutation of the major PKC phosphorylation in TRP site results in a slower deactivation kinetics [166].…”

Phototransduction in Drosophila

“…INAC has been shown to play critical roles in deactivating TRP channels [61]. INAC is one member of the PKC family of serine/threonine protein kinases [62,63], which is activated by Ca 2+ and DAG [64].…”

“…INAC is one member of the PKC family of serine/threonine protein kinases [62,63], which is activated by Ca 2+ and DAG [64]. Using phosphorylation assays in isolated signaling complexes, TRP was identified to undergo Ca 2+ -dependent phosphorylation modification [61]. CaM is likely to be another regulator in the deactivation of TRP and TRPL channels.…”

Calcium Transients in the Rhabdomeres of Dark- and Light-Adapted Fly Photoreceptor Cells

“…Ca 2ϩ has been proposed to act directly on the light-activated channels (Hardie and Minke, 1994;Hardie, 1995;Obukhov et al, 1998), or via binding to calmodulin that interacts with the light-activated channels (Phillips et al, 1992: Warr andKelly, 1996;Scott et al, 1997). Additionally, a Ca 2ϩ -regulated PKC (Huber et al, 1998) is crucial for Ca 2ϩ -dependent deactivation of the light response (Ranganathan et al, 1991) and light adaptation (Hardie et al, 1993). Other important molecules in the phototransduction cascade that have been suggested to be modulated by Ca 2ϩ or by Ca 2ϩ /calmodulin include neitherinactivation-nor-afterpotential C (Porter et al, 1993), inactivationnor-afterpotential D (Chevesich et al, 1997), and PLC (Running Deer et al, 1995).…”