Essential Role for Protein Kinase D Family Kinases in the Regulation of Class II Histone Deacetylases in B Lymphocytes

Abstract

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.The protein kinase D (PKD) family comprises three different but closely related serine kinases, PKD1, PKD2, and PKD3, all of which have a highly conserved N-terminal regulatory domain containing two cysteine-rich diacylglycerol (DAG) binding domains and an autoinhibitory pleckstrin homology (PH) domain. PKD enzymes are highly expressed in hematopoietic cells, and they are selectively activated by the engagement of antigen receptors, including the B-cell antigen receptor (BCR), the T-cell receptor, and the FcεR1 in B cells, T cells, and mast cells, respectively (22,24). PKD family members are activated by a signaling pathway involving gamma phospholipase C activation, production of diacylglycerol, and activation of classical/novel PKCs. PKC-mediated phosphorylation of two conserved serine residues in the catalytic domains of PKDs is essential for their activation (13,22,45,48,49). In addition, binding of DAG to the regulatory domain of PKD contributes both to PKD1 activation (50) and to PKD subcellular localization (21,23,(38)(39)(40).PKD enzymes are predicted to play important functions in controlling lymphocyte biology, but most of the evidence that supports this hypothesis is indirect. For example, studies in transgenic mice have shown that constitutively active PKD1 can substitute for the pre-T-cell antigen receptor complex to regulate early thymocyte differentiation and proliferation (20). In other cell types, PKD enzymes have also been implicated in the regulation of Golgi organization and protein trafficking to the cell surface (1,8,12,14,18,53), cell survival (44), 43,44), glucose transport (5), and integrin activation/recycling (29, 51).In addition, it has been ...

Protein Kinase D3 Is a Pivotal Activator of Pathological Cardiac Hypertrophy by Selectively Increasing the Expression of Hypertrophic Transcription Factors

“…Unexpectedly, a recent study reported a newly developed PKD1-selective inhibitor failed to attenuate PCH in whole animal models (31). One possible explanation for such a discrepancy is that PKD3 could substitute for PKD1 as a HDAC5 kinase as documented in non-cardiac cells (32). Our new results shown herein indicate PKD3 is indeed required for PCH but works through a different mechanism.…”

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

“…[37][38][39][40] To extend results from T lymphocytes, we studied exosome secretion in DT40 chicken B lymphocytes that lacked their two PKD isotypes, PKD1 and PKD3. 36 Wild-type and PKD1 − / − 3 − / − DT40 cells were transfected with GFP-CD63 and exosome secretion was assessed after stimulation with PMA plus ionomycin or with anti-IgM. PMA plus ionomycin induced exosome secretion in WT, but not in .…”

Protein Kinase D Isoforms Are Activated in an Agonist-specific Manner in Cardiomyocytes

“…While this molecular heterogeneity is generally assumed to reflect the co-expression of multiple PKD isoforms with distinct cellular functions, direct experimental evidence that native PKD isoforms are activated in a stimulusspecific manner and/or phosphorylate different target substrates in vivo in a cellular context has never been published. In fact, studies using either RNA interference technology or genetic knock-out strategies generally identify functionally redundancy for individual PKD isoforms (2,10). This study is the first to show that PKD1 and PKD2 are activated in an agonist-specific manner in cardiomyocytes.…”