Purification and characterization of ceramide-activated protein phosphatases.
Ceramide has emerged as a potential regulator of diverse cellular functions, and a few direct targets have been identified for its action including protein kinases and phosphatases. In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purified to near homogeneity using hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS-polyacrylamide gel electrophoresis which comigrated with the three subunits of heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB'C and ABalphaC as well as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by the coelution of CAPP with trimeric and dimeric PP2A on size-exclusion chromatography. These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the biochemical investigation of CAPP.
Galadari
S
S
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kishikawa
K
K
Kamibayashi
C
C
Mumby
M C
MC
Hannun
Y A
YA
D000818:Animals; D015153:Blotting, Western; D001921:Brain; D002518:Ceramides / Q000502:physiology*; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification*; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology
D002518:Ceramides; C064769:N-acetylsphingosine; C033452:Phenyl-Sepharose CL-4B; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; D013110:Sphingosine