Reversible Phosphorylation of the Signal Transduction Complex in Drosophila Photoreceptors

Abstract

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase C␤ (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. Protein kinase C (PKC)1 modulates various biological processes by transferring phosphates to serine and threonine residues of substrates. Regulatory proteins such as RACK (receptor for activated PKC) and RICK (receptor for inactive PKC) have been shown to target PKC to specific subcellular compartments (1). This selective distribution of the kinase directs its catalytic activity toward restricted groups of substrates localized proximal to the enzyme. The Drosophila visual cascade is a G q -coupled phospholipase C␤ (PLC␤)-mediated pathway (see Ref. 2 for review). In this cascade, an eye-specific isoform of PKC (eye-PKC) negatively regulates visual signaling (3, 4). Eye-PKC is localized to the rhabdomere (4), a densely packed microvillar structure where visual transduction takes place, by tethering to the adaptor protein INAD (inactivation-no-afterpotential D) (5).INAD contains five distinct PDZ (Postsynaptic density 95; Drosophila Discs large; and Zonula occludens 1) domains (5-8). PDZ domains are modular protein-protein interaction sequences consisting of 90 -100 amino acids. Proteins containing PDZ domains have been implicated in clustering signaling molecules, including enzymes, receptors, and channels (9). PDZ domains mediate at least three types of protein-protein associations. The most common interaction is the binding of a PDZ domain to a tetrapeptide motif, X-S/T-X--COOH (X, any amino acid; , hydrophobic residues), at the carboxyl terminus of target proteins (9, 10). The second type of interaction ...

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].…”

Rescue of Light Responses in the Drosophila “Null” Phospholipase C Mutant, norpA, by the Diacylglycerol Kinase Mutant, rdgA, and by Metabolic Inhibition

“…8). Furthermore, rhodopsin, arrestin, the PDZ domain-scaffolding molecule INAD, and the TRP channels are all phosphorylated during the light response (32)(33)(34)(35); although with the exception of arrestin (36), the functions of these phosphorylations remain obscure. We have in fact found one situation in which metabolic inhibitors appear to activate the light-sensitive channels independently of DAG.…”

Role of Protein Phosphatase 2A in Regulating the Visual Signaling inDrosophila

“…Previously, we had shown that protein phosphatases in the cytosolic extracts of wild-type fly heads could dephosphorylate INAD (Liu et al, 2000), and the critical protein phosphatases appeared to be enriched in photoreceptors. Because most of protein phosphatases seem to be ubiquitously expressed, we reinvestigated whether dephosphorylation of INAD is indeed mediated by protein phosphatases specifically expressed in photoreceptors by examining extracts prepared from glass flies that lack photoreceptors (Moses et al, 1989).…”

“…At the molecular level, eye-PKC is tethered to a macromolecular complex by interacting with inactivation no afterpotential D (INAD), which is a scaffolding protein that organizes the formation of the protein complex by colocalizing NORPA, TRP, and eye-PKC (Huber et al, 1996b;Tsunoda et al, 1997;Adamski et al, 1998;Li and Montell, 2000). Eye-PKC has been shown to phosphorylate INAD and TRP (Huber et al, 1996a(Huber et al, , 1998Liu et al, 2000;Popescu et al, 2006). Recently, we demonstrated that eye-PKC phosphorylates TRP at Ser 982 in vivo, and a loss of phosphorylation in TRP leads to slow deactivation of the visual response (Popescu et al, 2006).…”