Mcl-1 degradation during hepatocyte lipoapoptosis.
The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCdelta and PKC, only activation of PKC was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKC polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKC reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkc also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser(159), a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKC-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.
Masuoka
Howard C
HC
Division of Gastroenterology and Hepatology, College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Mott
Justin
J
Bronk
Steven F
SF
Werneburg
Nathan W
NW
Akazawa
Yuko
Y
Kaufmann
Scott H
SH
Gores
Gregory J
GJ
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D005227:Fatty Acids / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010168:Palmitates / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013228:Stearates / Q000494:pharmacology
D005227:Fatty Acids; C579094:Mcl1 protein, mouse; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010168:Palmitates; D019253:Proto-Oncogene Proteins c-bcl-2; D013228:Stearates; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex