Richard T. Waldron

Protein Kinase D Signaling

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as one of the fundamental mechanisms of signal transduction in animal cells. A plethora of external signals, including hormones, neurotransmitters, growth factors, cytokines, bioactive lipids, and tastants promote the stimulation of the isoforms of the PLC family, including , , , and . PLCs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce two second messengers: inositol 1,4,5-P3, which triggers the release of Ca from internal stores, and DAG, which elicits cellular responses through a variety of effectors (1). The most prominent intracellular targets of DAG are the classic ( , , ) and novel ( , , , ) isoforms of PKC, which are differentially expressed in cells and tissues (2, 3). The mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely understood. Protein kinase D (PKD), the founding member of a new family of serine/threonine protein kinases and the subject of this minireview, occupies a unique position in the signal transduction pathways initiated by DAG and PKC. PKD not only is a direct DAG target but also lies downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes.

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates 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. Immunoprecipi tation 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‐immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full‐length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c‐Jun as well as p53. CK2 phosphoryl ates 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.

Identification of in Vivo Phosphorylation Sites Required for Protein Kinase D Activation

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 Ser744 and Ser748 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 32P-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 Ser744 and Ser748 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.

The RAS Effector RIN1 Directly Competes with RAF and Is Regulated by 14-3-3 Proteins

ABSTRACT Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K d , 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1S351A, showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCμ]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.

The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C

The results presented here demonstrate that protein kinase D (PKD) and PKCη transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCη. The presence of PKCη in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCη was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCε, and did not bind PKCζ. When PKCη was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCη immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCη. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCη. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs ε and η, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.

Sequential Protein Kinase C (PKC)-dependent and PKC-independent Protein Kinase D Catalytic Activation via Gq-coupled Receptors DIFFERENTIAL REGULATION OF ACTIVATION LOOP SER744 AND SER748 PHOSPHORYLATION

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser744 and Ser748 revealed striking PKC-independent phosphorylation of Ser748 as well as Ser744 phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser744 and Ser748 is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser748 is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.

Protein kinase D complexes with C-Jun N-terminal kinase via activation loop phosphorylation and phosphorylates the C-Jun N-terminus.

Protein kinase D (PKD), a downstream effector of protein kinase C (PKC), is implicated in suppression of the c-Jun N-terminal kinase (JNK) signaling pathway, however, its mechanism of action is unclear. Transphosphorylation of the PKD activation loop at serines 744/748 by a PKC mediated signal transduction pathway enhances its catalytic activity. Here we show that PKD activation loop phosphorylation at serines 744/748 via PKC, or mutation of these serines to glutamic acid (PKD-S744/748E) also results in complex formation with JNK, indicating that suppression of JNK signaling by PKD involves a direct interaction with JNK. Because catalytically active PKD associates with JNK we determined whether it could phosphorylate the c-Jun N-terminus as a potential mechanism by which it suppresses c-Jun Ser 63 phosphorylation when it complexes with JNK. Purified human PKD and either wild-type PKD from phorbol 12, 13-dibutyrate (PDB)-stimulated cells or unstimulated constitutively active PKD (PKD-S744/748E), phosphorylated the c-Jun N-terminus between amino acids 1–89 at sites distinct from those phosphorylated by JNK. These results demonstrate, for the first time, phosphorylation dependent association of PKD with another signaling molecule and reveal a potential mechanism by which PKD could modulate the ability of JNK to phosphorylate c-Jun by phosphorylating alternative sites in the c-Jun N-terminus when it is complexed with JNK.

Phosphorylation-dependent protein kinase Dactivation

The novel mouse serine‐threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides and growth factors via a phosphorylation‐dependent mechanism requiring protein kinase C (PKC) activity. Structural comparison of the PKD catalytic domain with other kinases reveals a close similarity with MEK familiy kinases, which are activated upon phosphorylation of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in which putative activation loop serine residues 744 and 748 were mutated to either alanine or glutamic acid into COS‐7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form upon phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid replacement mutants were partially activated. In vivo 32P‐labeling and two‐dimensional phosphopeptide mapping of PKD and catalytically inactive PKD mutants at serine 744, 748 or at both residues revealed that phorbol ester‐sensitive phosphopeptides could be selectively eliminated from pattrens observed as a result of these mutations. Treatment of cells with the PKC inhibitor GFI also prevented the appearance of phosphopeptide spots occuring in response to phorbol ester stimulation. These results provide direct evidence that PKD becomes activated in vivo as a consequence of PKC‐mediated phosphorylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different Gq-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser744 and Ser748. Transphosphorylation targeted Ser744, whereas autophosphorylation was the predominant mechanism for Ser748 in cells stimulated with Gq-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to Gq-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of Gq-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.

Effects of Oxidative Alcohol Metabolism on the Mitochondrial Permeability Transition Pore and Necrosis in a Mouse Model of Alcoholic Pancreatitis

BACKGROUND & AIMS Opening of the mitochondrial permeability transition pore (MPTP) causes loss of the mitochondrial membrane potential (ΔΨm) and, ultimately, adenosine triphosphate depletion and necrosis. Cells deficient in cyclophilin D (CypD), a component of the MPTP, are resistant to MPTP opening, loss of ΔΨm, and necrosis. Alcohol abuse is a major risk factor for pancreatitis and is believed to sensitize the pancreas to stressors, by poorly understood mechanisms. We investigated the effects of ethanol on the pancreatic MPTP, the mechanisms of these effects, and their role in pancreatitis. METHODS We measured ΔΨm in mouse pancreatic acinar cells incubated with ethanol alone and in combination with physiologic and pathologic concentrations of cholecystokinin-8 (CCK). To examine the role of MPTP, we used ex vivo and in vivo models of pancreatitis, induced in wild-type and CypD(-/-) mice by a combination of ethanol and CCK. RESULTS Ethanol reduced basal ΔΨm and converted a transient depolarization, induced by physiologic concentrations of CCK, into a sustained decrease in ΔΨm, resulting in reduced cellular adenosine triphosphate and increased necrosis. The effects of ethanol and CCK were mediated by MPTP because they were not observed in CypD(-/-) acinar cells. Ethanol and CCK activated MPTP through different mechanisms-ethanol by reducing the ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, as a result of oxidative metabolism, and CCK by increasing cytosolic Ca(2+). CypD(-/-) mice developed a less-severe form of pancreatitis after administration of ethanol and CCK. CONCLUSIONS Oxidative metabolism of ethanol sensitizes pancreatic mitochondria to activate MPTP, leading to mitochondrial failure; this makes the pancreas susceptible to necrotizing pancreatitis.