Protein Kinase D Directly Phosphorylates Ser498 of Histone Deacetylase 5 via a Random Sequential Kinetic Mechanism

Abstract

Class II histone deacetylases (HDACs) are signal-responsive repressors of gene transcription. In the heart, class II HDAC 5 suppresses expression of genes that govern stress-induced cardiomyocyte growth. Signaling via pro-growth G protein coupled receptors (GPCRs) triggers phosphorylation of HDAC5 on two serine residues (Ser259 and Ser498), resulting in nuclear export of HDAC5 and de-repression downstream target genes. Although prior studies established a role for protein kinase D (PKD) in the regulation of HDAC5 phosphorylation, it remained unclear whether PKD functions directly or indirectly to control the phosphorylation status of this transcriptional repressor. Here, we demonstrate that PKD catalyzes direct phosphoryl-group transfer to Ser498 of HDAC5. Each of the three PKD family members, PKD1, PKD2 and PKD3, is capable of phosphorylating HDAC5 (Km for substrate = 2.07, 3.12 and 1.43 uM, respectively), although PKD2 exhibits highest catalytic efficiency (kcat/Km = 6.77 min−1 uM−1). Kinetic studies revealed that the three PKD isozymes phosphorylate HDAC5 through a random sequential mechanism, and that ATP has no effect on association of kinase with peptide substrate. In addition, we demonstrate that ADP competitively inhibits phosphorylation of HDAC5 (Ki = 8.50, 17.54 and 11.98 uM for PKD1, PKD2 and PKD3, respectively), suggesting that PKD is subject to product feedback inhibition. These findings define PKD as an HDAC kinase and thus further support PKD as a key regulator of HDAC phosphorylation and nuclear export.