PHOSRESTIN-I UNDERGOES THE EARLIEST LIGHT-INDUCED PHOSPHORYLATION BY A CALCIUM CALMODULIN-DEPENDENT PROTEIN-KINASE IN DROSOPHILA PHOTORECEPTORS/

Activation of PI-PLC initiates two independent branches of protein pho sphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-depen dent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosoph ila homolog of vertebrate photoreceptor arrestin, undergoes light-indu ced phosphorylation on a subsecond time scale which is faster than tha t of any other protein in vivo. We determine that a CaMK activity is r esponsible for in vitro PRI phosphorylation at Ser(366) in the C-termi nal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) repr esents phosphoserine(366). We also demonstrate that Ser(366) is the ph osphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. Fro m these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event followin g activation of PI-PLC in living Drosophila photoreceptors.