Protein Kinase D Induces Transcription through Direct Phosphorylation of the cAMP-response Element-binding Protein

Abstract

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified. In this study we demonstrate that the transcription factor cAMPresponse element-binding protein (CREB) is a direct substrate for PKD. PKD1 and CREB interact in cells, and activated PKD1 provokes CREB phosphorylation at Ser-133 both in vitro and in vivo. A constitutive active mutant of PKD1 stimulates GAL4-CREB-mediated transcription in a Ser-133-dependent manner, activates CRE-responsive promoters, and increases the expression of CREB target genes. PKD1 also enhances transcription mediated by two other members of the CREB family, ATF-1 and CREM. Our results describe a novel mechanism for PKD-induced signaling through activation of the transcription factor CREB and suggest that stimulus-induced phosphorylation of CREB, reported to be mediated by protein kinase C, may involve downstream activated PKD.The mammalian PKD 3 family of serine/threonine kinases includes the isoforms PKD1 (mouse PKD and human PKC), PKD2, and PKD3 (also named PKC). PKD was originally considered to be a member of the PKC family (1, 2) but is now classified in the calcium/calmodulin-dependent kinase group based on sequence similarities in the kinase domain (3). The PKDs share a similar architecture consisting of a C-terminal catalytic domain, an N-terminal regulatory domain that encompasses two cysteine-rich regions (C1a and C1b), and a pleckstrin homology (PH) domain (4 -7). A comparison of the amino acid sequences of PKD1-3 reveals that the highest homology lies in the catalytic domain, followed by C1a, C1b, and PH domain, suggesting that isoform-specific functions may be due to the regulatory part of the kinase (8).PKD1 can be activated by several mechanisms. The most studied mechanism involves a sequential activation of phospholipase C that results in the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol (DAG) and subsequent activation of the classical (␣, ␤I, ␤II, and ␥) and novel (␦, ⑀, , and ) PKC isoforms. Binding of DAG to the C1b domain of PKD1 directs its translocation to the plasma membrane where activated novel PKC phosphorylates PKD1 at Ser-744/748 in the activation loop, causing activation of the enzyme. The activated enzyme can be imported via its C1b motif into the nucleus, where it transiently accumulates before being exported to the cytosol through a CRM1-dependent nuclear export pathway that requires the PH domain of PKD (4 -7). Mutations and deletions in the regulatory domain induce activation of PKD to various extents, and the entire regulatory domain has an inhibitory effect on the kinase activity (9). Indeed, PKD can also be activated through caspase-mediated cleavage of the regulatory domain (10). G␤␥ subunits ...

Activation of Protein Kinase D1 in Mast Cells in Response to Innate, Adaptive, and Growth Factor Signals

“…5), which may reflect an inhibitory effect of certain PKC. These data suggest that transcription and/or stabilization of CCL2 mRNA is downstream of PKD1 activation, consistent with reports in other cell types that activation of PKD1 fosters gene transcription through regulation of class II histone deacetylases and/or transcription factors (33)(34)(35)(36)(37)(38). The effects of PKD1 activation also extended to the release of CCL2 protein induced by Pam3CSK4, SCF, and cross-linked IgE, as they showed the identical pharmacologic profile measured 2 h after cell activation (Fig.…”

“…Particularly notable are the findings that phosphorylation of histone deacetylases 5 and 7 by PKD1 regulates their export from the nucleus, leading to derepression of the transcription of certain genes (33)(34)(35)(36)(37). In addition, PKD1 phosphorylates members of the cAMP-response elementbinding protein family of transcription factors, leading to increased transcriptional activity (38). PKD1 belongs to a family of three homologous enzymes (6,39,40) that can differ in subcellular localization (41)(42)(43) and function (29,37,44).…”

Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser744/Ser748 and Ser916

“…However, the list of known PKD substrates remains relatively short. We and others recently implicated PKD as a CREB-Ser 133 kinase that regulates Cre-dependent transcriptional responses (6,7). PKD also functions as a physiologically relevant HDAC5 kinase (8).…”

Protein kinase D in vascular biology and angiogenesis

“…PKD-mediated transcriptional control can occur through at least two mechanisms. PKD directly phosphorylates the transcription factor CREB to allow recruitment of coactivators, including CREB binding protein (CBP)/p300, and augment CREB-mediated gene expression (26). In a recent study, CREB dependent transcription of the immediate-early gene, Nurr1, was found to be important for in vivo angiogenesis in a mouse model using matrigel plugs (27).…”