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 language | English (US) |
|---|---|
| Pages (from-to) | 11549-11561 |
| Number of pages | 13 |
| Journal | Journal of Biological Chemistry |
| Volume | 282 |
| Issue number | 15 |
| DOIs | |
| State | Published - Apr 13 2007 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation. / Zeidan, Youssef; Hannun, Yusuf A.
In: Journal of Biological Chemistry, Vol. 282, No. 15, 13.04.2007, p. 11549-11561.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation
AU - Zeidan, Youssef
AU - Hannun, Yusuf A.
PY - 2007/4/13
Y1 - 2007/4/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=34249657837&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34249657837&partnerID=8YFLogxK
U2 - 10.1074/jbc.M609424200
DO - 10.1074/jbc.M609424200
M3 - Article
C2 - 17303575
AN - SCOPUS:34249657837
VL - 282
SP - 11549
EP - 11561
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -