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, transformation, and gene expression; however, identifying phosphorylation events that mediate these responses has been difficult. We expression-cloned a group of PKC-binding proteins and identified a high molecular weight, heat-soluble protein as the major PKC-binding protein in REF52 fibroblasts (Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C., and Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422). 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 spectrometry, serine residues 283, 300, 507, and 515 were identified as the major in vitro PKC phosphorylation sites in clone 72. Phosphorylation state-selective antibodies were raised against phosphopeptides encompassing each of the four phosphorylation sites. These antibodies were used to determine that phorbol esters stimulate phosphorylation of serines 283, 300, 507, and 515 in cultured cells, indicating that clone 72 is directly phosphorylated by PKC in living cells. Phosphorylated clone 72 preferentially accumulates in membrane protrusions and ruffles, indicating that PKC activation and clone 72 phosphorylation are involved in membrane-cytoskeleton remodeling. These data lend further evidence to the model that PKCs directly interact with, phosphorylate, and modify the functions of a group of substrate proteins, STICKs (substrates that interact with C-kinase).
Chapline
C
C
Adirondack Biomedical Research Institute, Lake Placid, New York 12946, USA.
Cottom
J
J
Tobin
H
H
Hulmes
J
J
Crabb
J
J
Jaken
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D007150:Immunohistochemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*
D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D010768:Phosphoserine; D011493:Protein Kinase C