The dimeric and catalytic subunit forms of protein phosphatase 2A from rat brain are stimulated by C2-ceramide.
Protein phosphatase 2A (PP-2A) is a heterotrimeric enzyme consisting of a catalytic (C) subunit and A and B regulatory subunits. PP-2A is activated by ceramide in vitro suggesting that PP-2A may be a target of this putative second messenger in vivo (Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530). In this study, sensitivity to ceramide was only observed when the B subunit was present, suggesting that the B subunit was required for ceramide activation. Here we show that dimeric PP-2A, produced from trimeric PP-2A by heparin-agarose-induced dissociation of the B subunit and isolated by preparative native electrophoresis, is activated by ceramide. The catalytic subunit of PP-2A, produced from trimeric PP-2A by freezing and thawing in the presence of 0.2 M beta-mercaptoethanol and isolated by gel filtration, is also activated by ceramide. The trimeric and catalytic subunit forms of PP-2A exhibit a similar dose dependence of activation by ceramide, and are stimulated to a similar extent at ceramide concentrations yielding maximal activation. These findings indicate that neither the A nor the B subunit is required for ceramide stimulation of PP-2A. Together, these results demonstrate that the catalytic subunit contains a ceramide binding site and suggest that efforts to understand the mechanism of activation of PP-2A by ceramide should be focused on this subunit. The discovery that the catalytic subunit contains a ceramide binding site raises the possibility that other members of this serine/threonine phosphatase gene family may contain lipid binding sites and be regulated by ceramide or other lipid second messengers.
Law
B
B
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Rossie
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S
D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D010749:Phosphoprotein Phosphatases / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D054648:Protein Phosphatase 2; D051381:Rats; D015290:Second Messenger Systems; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D007527:Isoenzymes; C064769:N-acetylsphingosine; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D013110:Sphingosine