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 phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser(366) in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine(366). We also demonstrate that Ser(366) is the phosphorylation 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. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.