PURIFICATION AND CHARACTERIZATION OF CERAMIDE-ACTIVATED PROTEIN PHOSPHATASES

Ceramide has emerged as a potential regulator of diverse cellular func tions, and a few direct targets have been identified for its action in cluding protein kinases and phosphatases. In this study, we have purif ied the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purif ied to near homogeneity using hydrophobic interaction chromatography o n phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS-polyacr ylamide gel electrophoresis which comigrated with the three subunits o f heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB'C and AB alpha C as wel l as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by th e coelution of CAPP with trimeric and dimeric PP2A on size-exclusion c hromatography. These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the bi ochemical investigation of CAPP.