title: Phosphorylation of Cdc42 effector protein-4 (CEP4) by protein kinase C promotes motility of human breast cells.
abstract: Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser(18), Ser(77), Ser(80), and Ser(86)) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser(18) and Ser(80) were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser(18) and Ser(80) causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.
authors.affiliation: From the Department of Chemistry and Biochemistry, Queens College, Flushing, New York 11367 and The Graduate Center, The City University of New York, New York, New York 10016.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: From the Department of Chemistry and Biochemistry, Queens College, Flushing, New York 11367 and Susan.Rotenberg@qc.cuny.edu.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Rotenberg
mesh_terms: D001940:Breast / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D003598:Cytoskeletal Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D020555:GTP-Binding Protein Regulators / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D016601:RNA-Binding Proteins; D015398:Signal Transduction / Q000235:genetics; D020741:rho GTP-Binding Proteins
chemical_list: C422985:CDC42EP4 protein, human; D003598:Cytoskeletal Proteins; D020555:GTP-Binding Protein Regulators; D016601:RNA-Binding Proteins; D011493:Protein Kinase C; D020741:rho GTP-Binding Proteins
keywords: Actin; Breast Cancer; GTPase; Guanine Nucleotide Exchange Factor (GEF); Protein Kinase C, Cdc42, Rac, Mutant; Protein Phosphorylation; Protein Purification
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