CaM kinase IIdeltaC phosphorylation of 14-3-3beta in vascular smooth muscle cells: activation of class II HDAC repression.
The myocyte enhancer factor-2 (MEF2) family of transcription factors regulates transcription of muscle-dependent genes in cardiac, skeletal and smooth muscle. They are activated by calcium/calmodulin (CaM)-dependent protein kinases I and IV and silenced by CaM KIIdeltaC. MEF2 is held in an inactive form by the class II histone deacetylases (HDAC) until phosphorylated by either CaM kinase I or IV. Upon phosphorylation, HDAC is transported out of the nucleus via a 14-3-3 dependent mechanism freeing MEF2 to drive transcription. The 14-3-3 chaperone protein exists as a homodimer. In the region of homodimerization, there are two canonical CaM kinase II phosphorylation sites (ser60 and ser65). In vitro phosphorylation assay results indicate that 14-3-3beta is indeed a substrate for CaM kinase II. We hypothesize that CaM kinase IIdeltaC phosphorylation of 14-3-3beta will disrupt homodimer formation resulting in the return of HDAC to the nucleus and their reassociation with MEF2. To test this, we mutated serines 60 and 65 of 14-3-3beta to aspartates to mimic the phosphorylated state. In MEF2 enhancer-reporter assays in smooth muscle cells, expression of the 14-3-3beta double mutant attenuated MEF2-enhancer activity driven by CaM kinase I or IV. The intracellular fate of HDAC4 was followed by transfection of smooth muscle cells with an HDAC4-Green Fluorescent Protein fusion hybrid. The 14-3-3beta double mutant prevented HDAC4 cytoplasmic localization in the presence of active CaM kinase I or IV. These data suggest that the mechanism of CaM kinase IIdeltaC silencing of MEF-2-dependent genes is by phosphorylation of 14-3-3beta, which allows HDAC to return to the nucleus to reform a complex with MEF2, thereby silencing MADS box-dependent gene induction in smooth muscle.
Ellis
Joel J
JJ
Laboratory of Cardiac and Vascular Molecular Genetics, Cardiovascular Research Institute and Department of Integrative Physiology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Valencia
Thomas G
TG
Zeng
Hong
H
Roberts
L Don
LD
Deaton
Rebecca A
RA
Grant
Stephen R
SR
D048948:14-3-3 Proteins; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D006655:Histone Deacetylases / Q000145:classification* / Q000378:metabolism*; D064326:MEF2 Transcription Factors; D008285:Major Histocompatibility Complex / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000201:enzymology / Q000378:metabolism*; D018006:Myogenic Regulatory Factors; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D012097:Repressor Proteins / Q000145:classification* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D002147:Calmodulin; D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D012097:Repressor Proteins; D014157:Transcription Factors; C482982:YWHAB protein, human; D010656:Phenylephrine; D014446:Tyrosine 3-Monooxygenase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C400300:HDAC4 protein, human; D006655:Histone Deacetylases