CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy
J. Clin. Invest. Johannes Backs, et al. 116:1853 doi:10.1172/JCI27438 [Go to this article.]

Figure 5
R601 of HDAC4 is required for full responsiveness to CaMKIIδB-T287D. (A and B) FLAG-HDAC4 mutants carrying point mutations in the CaMKII docking region were coexpressed with Myc-CaMKIIδB-T287D in COS cells. Coimmunoprecipitation revealed that substitution of R601 with either alanine or phenylalanine prevented a physical interaction with CaMKIIδB-T287D. (C and D) Myc-CaMKIIδB-T287D or CaMKI c.a. and either FLAG-HDAC4-WT, -R601A, or -R601F were coexpressed in COS cells, and HDAC4 localization was determined 1 day after transfection. HDAC4-R601A and -R601F were still responsive to CaMKI but not to CaMKIIδB-T287D and did not colocalize with the kinase. (C) Representative images. Magnification, ×40. (D) Quantitative analysis. (E) Mammalian 2-hybrid assay with GAL4-HDAC4 mutants and VP16–14-3-3. Substitution of R601 with phenylalanine and leucine prevented and with alanine and lysine markedly attenuated 14-3-3 binding. (F) In vitro kinase assay. Active His-CaMKIIδ induced phosphorylation of GST-HDAC4-WT (amino acids 419–670) but not of the GST-HDAC4-R601F (419–670) mutant. Ca²⁺-depletion by EGTA prevented HDAC4 phosphorylation by CaMKII. (G) GST pull-down assay with GST, GST-HDAC4-WT (419–670), GST-HDAC4-R601F (419–670) mutant, and active His-CaMKIIδ. (H) Coimmunoprecipitation revealed that HA-CaMKI docks to FLAG-HDAC4-WT and -R601F to the same degree, however, it docks to HDAC5 with greater affinity.