Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells [Cell Biology]

August 1st, 2014 by Zhao, X., Rotenberg, S. A.

Cdc42 effector protein-4 (CEP4) was recently identified by this 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 (Ser18, Ser77, Ser80, and Ser86) were individually replaced with Asp (D) 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 Ser18 and Ser80 were directly phosphorylated by PKCĪ± 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 TAP vector, followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as 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 Ser18 and Ser80 causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodia formation, and cell motility.