Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility.
Coronins are a conserved family of WD repeat-containing, actin-binding proteins that regulate cell motility in a variety of model organisms. Our results show that Coronin 1B is a ubiquitously expressed member of the mammalian Coronin gene family that co-localizes with the Arp2/3 complex at the leading edge of fibroblasts, and co-immunoprecipitates with this complex. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated by protein kinase C (PKC) phosphorylation. Coronin 1B is phosphorylated by PKC both in vitro and in vivo. Using tryptic peptide mapping and mutagenesis, we have identified serine 2 (Ser-2) on Coronin 1B as the major residue phosphorylated by PKC in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to phorbol 12-myristate 13-acetate (PMA) and increased speed in single cell tracking assays. Cells expressing the Coronin 1B S2D mutant have attenuated PMA-induced ruffling and slower cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed, whereas expression of the S2D mutant renders cells hypersensitive to its effects. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts, and that its ability to control motility and interactions with the Arp2/3 complex are regulated by PKC phosphorylation at Ser-2. Furthermore, Coronin 1B phosphorylation is responsible for a significant fraction of the effects of PMA on fibroblast motility.
Cai
Liang
L
Lineberger Comprehensive Cancer Center and Department of Cell & Developmental Biology, University of North Carolina Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, USA.
Holoweckyj
Nicholas
N
Schaller
Michael D
MD
Bear
James E
JE
D016475:3T3 Cells; D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism; D014161:Transduction, Genetic; D054378:Acetogenins
D015107:4-Butyrolactone; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D003598:Cytoskeletal Proteins; D011493:Protein Kinase C; D012694:Serine; C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; C495927:Actr2 protein, mouse; C495929:Actr3 protein, mouse; C438920:coronin acetogenin; D054378:Acetogenins