Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors

In the Drosophila visual cascade, the transient receptor potential (TRP) ca lcium channel, phospholipase C beta (no-receptor-potential A), and an eye-s pecific isoform of protein kinase C (eye-PKC) comprise a multimolecular sig naling complex via their interaction with the scaffold protein INAD, Previo usly, we showed that the interaction between INAD and eye-PKC is a prerequi site for deactivation of a light response, suggesting eye-PKC phosphorylate s proteins in the complex. To identify substrates of eye-PKC, we immunoprec ipitated the complex from head lysates using anti-INAD antibodies and perfo rmed in vitro kinase assays. Wild-type immunocomplexes incubated with [P-32 ]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 okadai c acid, microcystin, and protein phosphatase inhibitor-2, These phosphatase activities were enriched in the cytosol of mild-type heads, but drasticall y reduced in extracts prepared from glass mutants, which lack photoreceptor s, Our findings indicate that INAD functions as RAGE (receptor for activate d PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephos phorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferent ially expressed in photoreceptor cells.