No abstract

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser 744 and Ser 748 with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from 32 P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/ S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser 744 and Ser 748 and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.Protein kinase C (PKC), 1 a major target for the tumor promoting phorbol esters, has been implicated in the signal transduction of a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation (1-5). Molecular cloning has demonstrated the presence of multiple related PKC isoforms (5-8) i.e. classic PKCs (␣, ␤1, ␤2, and ␥), novel PKCs (␦, ⑀, , and ) and atypical PKCs ( and ) all of which possess a highly conserved catalytic domain. Despite intense investigation, the events occurring downstream of specific isoforms of PKC remain poorly defined.The newly identified PKD is a mouse serine/threonine protein kinase with distinct structural and enzymological properties (9). The catalytic domain of PKD is distantly related to Ca 2ϩ -regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (10). Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs, indicating that PKD is a protein kinase with distinct substrate specificity (9, 11). In contrast to all known PKCs, including mammalian, Drosophila, and yeast isoforms (12), the NH 2 -terminal region of PKD contains a pleckstrin homology domain that regulates enzyme activity (13) and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif (9). However, the amino-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that binds phorbol esters with high affinity (9). Immunopurified PKD is markedly stimulated by either biologically active...

The COP9 signalosome (CSN) puri®ed from human erythrocytes possesses kinase activity that phosphorylates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immunoprecipitates with CSN from HeLa cells. CK2 binds to DCSN3(111±403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modi®es CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphorylates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun±Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is 744 and Ser 748 phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domainmediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-⌬PH was not abrogated by mutation of Ser 744 and Ser 748 to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKC⑀-PKD phosphorylation cascade and provide additional insight into the activation mechanism.

A close correlation was observed between intracellular Ca2+ pool depletion and refiling and the onset of DNA synthesis and proliferation of DDT,MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butylhydroquinone (DBHQ) and thapsigargin (TG) specificafly emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that foUlowing release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a Go-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for Go to G, transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.