Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors.
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.
Liu
M
M
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37232-6600, USA.
Parker
L L
LL
Wadzinski
B E
BE
Shieh
B H
BH
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005136:Eye Proteins / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D050051:Transient Receptor Potential Channels
D015220:Calcium Channels; D029721:Drosophila Proteins; D005136:Eye Proteins; D019476:Insect Proteins; D050051:Transient Receptor Potential Channels; C091568:inaD protein, Drosophila; C063169:trp protein, Drosophila; D008345:Manganese; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; D002118:Calcium