Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation

Youssef H. Zeidan, Yusuf A. Hannun

Research output: Contribution to journalArticle

118 Scopus citations

Abstract

Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCδ, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCδ-ASMase complex after PMA stimulation, and PKCδ was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser508 proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase S508A blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCδ serves as a key upstream kinase.

Original languageEnglish (US)
Pages (from-to)11549-11561
Number of pages13
JournalJournal of Biological Chemistry
Volume282
Issue number15
DOIs
StatePublished - Apr 13 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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