Protein kinase D: an intracellular traffic regulator on the move

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.     

WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.     

Integrin-linked kinase is a positive mediator of L6 myoblast differentiation

Overexpression of ILK in L6 myoblasts results in increased ILK kinase activity, stimulating myotube formation and induction of biochemical differentiation markers. Expression of a dominant negative ILK mutant, ILK(E359K), inhibits endogenous ILK activation and L6 differentiation. Cell cycle analysis of ILK(E359K) cells cultured in serum-free conditions indicates significant apoptosis (11-19% sub-diploid peak) which is not seen in insulin treated cells. Expression of ILK variants does not have significant effects on S-phase transit, however. Known targets of ILK, PKB/Akt or glycogen synthase kinase 3beta are not obviously involved in ILK-induced L6 differentiation. Insulin-stimulated phosphorylation of PKB at Ser473 is unimpaired in the ILK(E359K) cells, suggesting that PKB is not a myogenic target of ILK. Inhibition of GSK3beta by LiCl blocks L6 myogenesis, indicating that ILK-mediated inhibition of GSK3beta is not sufficient for differentiation. Our data do suggest that a LiCl-sensitive interaction of ILK is important in L6 myoblast differentiation.     

Protein kinase C theta is not essential for T-cell-mediated clearance of murine gammaherpesvirus 68

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. T cells are essential for primary clearance of MHV-68 and survival of mice following intranasal infection. Previous reports have suggested that protein kinase C theta (PKCtheta) is essential for T-cell activation and cytokine production in vitro. To determine the role of this molecule in vivo during the immune response to a viral infection, PKCtheta-/- mice were infected with MHV-68. Despite the essential role of T cells in viral clearance, PKCtheta-/- mice survived infection, cleared lytic virus, and maintained effective long-term control of latency. CD8 T-cell expansion, trafficking to the lung, and cytotoxic activity were similar in PKCtheta+/+ and PKCtheta-/- mice, whereas antiviral antibody and T-helper cell cytokine production were significantly lower in PKCtheta-/- mice than in PKCtheta+/+ mice. These studies demonstrate a differential requirement for PKCtheta in the immune response to MHV-68 and show that PKCtheta is not essential for the T-cell activation events leading to viral clearance.     

RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.     

Protein kinase Czeta is a negative regulator of protein kinase B activity

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.     

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.     

Protein kinase CK2 is an inhibitor of the neuronal Cdk5 kinase

The complex of Cdk5 and its neuronal activator p35 is a proline-directed Ser/Thr kinase that plays an important role in various neuronal functions. Deregulation of the Cdk5 enzymatic activity was found to associate with a number of neurodegenerative diseases. To search for regulatory factors of Cdk5-p35 in the brain, we developed biochemical affinity isolation using a recombinant protein comprising the N-terminal 149 amino acids of p35. The catalytic alpha-subunit of protein kinase CK2 (formerly known as casein kinase 2) was identified by mass spectrometry from the isolation. The association of CK2 with p35 and Cdk5 was demonstrated, and the CK2-binding sites were delineated in p35. Furthermore, CK2 displayed strong inhibition toward the Cdk5 activation by p35. The Cdk5 inhibition is dissociated from the kinase function of CK2 because the kinase-dead mutant of CK2 displayed the similar Cdk5 inhibitory activity as the wild-type enzyme. Further characterization showed that CK2 blocks the complex formation of Cdk5 and p35. Together, these findings suggest that CK2 acts as an inhibitor of Cdk5 in the brain.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation

Signaling by bone morphogenetic proteins (Bmps) plays a pivotal role in developmental and pathological processes, and is regulated by a complex interplay with secreted Bmp binding factors, including Crossveinless 2 (Cvl2). Although structurally related to the Bmp antagonist Chordin, Crossveinless 2 has been described to be both a Bmp agonist and antagonist. Here, we present the first loss-of-function study of a vertebrate cvl2 homologue, showing that zebrafish cvl2 is required in a positive feedback loop to promote Bmp signaling during embryonic dorsoventral patterning. In vivo, Cvl2 protein undergoes proteolytic cleavage and this cleavage converts Cvl2 from an anti- to a pro-Bmp factor. Embryonic epistasis analyses and protein interaction assays indicate that the pro-Bmp function of Cvl2 is partly accomplished by competing with Chordin for binding to Bmps. Studies in cell culture and embryos further suggest that the anti-Bmp effect of uncleaved Cvl2 is due to its association with the extracellular matrix, which is not found for cleaved Cvl2. Our data identify Cvl2 as an essential pro-Bmp factor during zebrafish embryogenesis, emphasizing the functional diversity of Bmp binding CR-domain proteins. Differential proteolytic processing as a mode of regulation might account for anti-Bmp effects in other contexts.     

The farnesoid X-receptor is an essential regulator of cholesterol homeostasis

To address the importance of the farnesoid X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-deficient (-/-) mouse model. Compared with wild-type, FXR(-/-) mice have increased plasma high density lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL cholesterol ester clearance. Concomitantly, FXR(-/-) mice exhibit reduced expression of hepatic genes involved in reverse cholesterol transport, most notably, that for scavenger receptor BI. FXR(-/-) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(-/-) mice, despite decreased expression of hepatic genes thought to be involved in this process. These data demonstrate that FXR is a critical regulator of normal cholesterol metabolism and that genetic changes affecting FXR function have the potential to be pro-atherogenic.     

Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK.