A MAJOR, TRANSFORMATION-SENSITIVE PKC-BINDING PROTEIN IS ALSO A PKC SUBSTRATE INVOLVED IN CYTOSKELETAL REMODELING

Protein kinase C (PKC) plays a major role in regulating cell growth, t ransformation, and gene expression; however, identifying phosphorylati on events that mediate these responses has been difficult. We expressi on-cloned a group of PKC-binding proteins and identified a high molecu lar weight, heat-soluble protein as the major PKC-binding protein in R EF52 fibroblasts (Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., L i, Y., Kiley, S. C., and Jaken, S. (1996) J. Biol. Chem. 271, 6417-642 2). In this study, we demonstrate that this PKC-binding protein, clone 72, is also a PKC substrate in vitro and in vivo. Using a combination of phosphopeptide mapping, Edman degradation, and electrospray mass s pectrometry, serine residues 283, 300, 507, and 515 were identified as the major in vitro PKC phosphorylation sites in clone 72. Phosphoryla tion state-selective antibodies were raised against phosphopeptides en compassing each of the four phosphorylation sites. These antibodies we re used to determine that phorbol esters stimulate phosphorylation of serines 283, 300, 507, and 515 in cultured cells, indicating that clon e 72 is directly phosphorylated by PKC in living cells. Phosphorylated clone 72 preferentially accumulates in membrane protrusions and ruffl es, indicating that PKC activation and clone 72 phosphorylation are in volved in membrane-cytoskeleton remodeling. These data lend further ev idence to the model that PKCs directly interact with, phosphorylate, a nd modify the functions of a group of substrate proteins, STICKs (subs trates that interact with C-kinase).