Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation

Youssef Zeidan, Yusuf A. Hannun

Research output: Contribution to journalArticle

114 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Sphingomyelin Phosphodiesterase
Phosphorylation
Protein Kinase C
Acetates
Chemical activation
Acids
Ceramides
Ultraviolet radiation
Serine
Acetate Kinase
Radiation
Mutagenesis
Sphingomyelins
RNA Interference
Site-Directed Mutagenesis
phorbol-12-myristate
Immunoprecipitation
Phosphotransferases
Experiments
Cells

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 journalArticle

Zeidan, Youssef ; Hannun, Yusuf A. / Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 15. pp. 11549-11561.
@article{0f5083c5da4547c39038fc5e8aa522f1,
title = "Activation of acid sphingomyelinase by protein kinase Cδ-mediated phosphorylation",
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.",
author = "Youssef Zeidan and Hannun, {Yusuf A.}",
year = "2007",
month = "4",
day = "13",
doi = "10.1074/jbc.M609424200",
language = "English (US)",
volume = "282",
pages = "11549--11561",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "15",

}

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 -