Trophoblast Cell Fusion and Differentiation Are Mediated by Both the Protein Kinase C and A Pathways

The syncytiotrophoblast of the human placenta is an epithelial barrier that interacts with maternal blood and is a key for the transfer of nutrients and other solutes to the developing fetus. The syncytiotrophoblast is a true syncytium and fusion of progenitor cytotrophoblasts is the cardinal event leading to the formation of this layer. BeWo cells are often used as a surrogate for cytotrophoblasts, since they can be induced to fuse, and then express certain differentiation markers associated with trophoblast syncytialization. Dysferlin, a syncytiotrophoblast membrane repair protein, is up-regulated in BeWo cells induced to fuse by treatment with forskolin; this fusion is thought to occur through cAMP/protein kinase A-dependent mechanisms. We hypothesized that dysferlin may also be up-regulated in response to fusion through other pathways. Here, we show that BeWo cells can also be induced to fuse by treatment with an activator of protein kinase C, and that this fusion is accompanied by increased expression of dysferlin. Moreover, a dramatic synergistic increase in dysferlin expression is observed when both the protein kinase A and protein kinase C pathways are activated in BeWo cells. This synergy in fusion is also accompanied by dramatic increases in mRNA for the placental fusion proteins syncytin 1, syncytin 2, as well as dysferlin. Dysferlin, however, was shown to be dispensable for stimulus-induced BeWo cell syncytialization, since dysferlin knockdown lines fused to the same extent as control cells. The classical trophoblast differentiation marker human chorionic gonadotropin was also monitored and changes in the expression closely parallel that of dysferlin in all of the experimental conditions employed. Thus different biochemical markers of trophoblast fusion behave in concert supporting the hypothesis that activation of both protein kinase C and A pathways lead to trophoblastic differentiation.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

蛋白激酶与细胞分化—两种分化诱导剂对酪氨酸蛋白激酶的抑制

在报道视黄酸(RA)是人肝癌细胞株SMMC-7721分化诱导剂的基础上,本文继续报道8-溴-环磷酸腺苷对该细胞也有分化诱导作用,两者都抑制细胞的增殖,降低γ-谷氨酰转肽酶(γ-GT)比活力和升高(ALP)碱性磷酸酶的比活力。在10μmol/LRA和0.5mmol/L-8-Br-cAMP处理细胞1、3、5天后,胞液和膜性组分中的酪氨酸蛋白激酶(TPK)的比活力均降低,其中RA对胞液TPK的作用在早期较明显,约降低30％,而对膜性TPK的影响则随培养天数而逐渐增加,至第5天下降达50％以上。8Br-cAMP则相反,对胞浆TPK的抑制主要发生在3天以后;约抑制43—53％,而对膜性组分则抑制率逐日降低,在第一天较为明显。因TPK是一个细胞增殖恶变的标志,故RA和8-溴-cAMP对TPK的抑制进一步证明这两种分化诱导剂对SMMC-7721细胞的逆转作用。     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Atypical Protein Kinase C Zeta: Potential Player in Cell Survival and Cell Migration of Ovarian Cancer

Ovarian cancer is one of the most aggressive gynaecological cancers, thus understanding the different biological pathways involved in ovarian cancer progression is important in identifying potential therapeutic targets for the disease. The aim of this study was to investigate the potential roles of Protein Kinase C Zeta (PRKCZ) in ovarian cancer. The atypical protein kinase C isoform, PRKCZ, is involved in the control of various signalling processes including cell proliferation, cell survival, and cell motility, all of which are important for cancer development and progression. Herein, we observe a significant increase in cell survival upon PRKCZ over-expression in SKOV3 ovarian cancer cells; additionally, when the cells are treated with small interference RNA (siRNA) targeting PRKCZ, the motility of SKOV3 cells decreased. Furthermore, we demonstrate that over-expression of PRKCZ results in gene and/or protein expression alterations of insulin-like growth factor 1 receptor (IGF1R) and integrin beta 3 (ITGB3) in SKOV3 and OVCAR3 cells. Collectively, our study describes PRKCZ as a potential regulatory component of the IGF1R and ITGB3 pathways and suggests that it may play critical roles in ovarian tumourigenesis.     

Differential Effects of Protein Kinase A on Ras Effector Pathways

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, Ras^S35 and Ras^G37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing Ras^S35, a mutant which binds Raf, or Ras^G37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. Ras^S35 stimulated morphological transformation and anchorage-independent growth. Ras^G37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in Ras^S35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing Ras^G37. The Ras mutants, in turn, exerted differential effects on TSH signaling. Ras^S35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while Ras^G37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.     

Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while Ras^Asn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events.     

Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

Hepatitis C virus (HCV) is prevalent worldwide and has become a major cause of liver dysfunction and hepatocellular carcinoma. The high prevalence of HCV reflects the persistent nature of infection and the large frequency of cases that resist the current interferon (IFN)-based anti-HCV therapeutic regimens. HCV resistance to IFN has been attributed, in part, to the function of the viral nonstructural 5A (NS5A) protein. NS5A from IFN-resistant strains of HCV can repress the PKR protein kinase, a mediator of the IFN-induced antiviral and apoptotic responses of the host cell and a tumor suppressor. Here we examined the relationship between HCV persistence and resistance to IFN therapy. When expressed in mammalian cells, NS5A from IFN-resistant HCV conferred IFN resistance to vesicular stomatitis virus (VSV), which normally is sensitive to the antiviral actions of IFN. NS5A blocked viral double-stranded RNA (dsRNA)-induced PKR activation and phosphorylation of eIF-2alpha in IFN-treated cells, resulting in high levels of VSV mRNA translation. Mutations within the PKR-binding domain of NS5A restored PKR function and the IFN-induced block to viral mRNA translation. The effects due to NS5A inhibition of PKR were not limited to the rescue of viral mRNA translation but also included a block in PKR-dependent host signaling pathways. Cells expressing NS5A exhibited defective PKR signaling and were refractory to apoptosis induced by exogenous dsRNA. Resistance to apoptosis was attributed to an NS5A-mediated block in eIF-2alpha phosphorylation. Moreover, cells expressing NS5A exhibited a transformed phenotype and formed solid tumors in vivo. Disruption of apoptosis and tumorogenesis required the PKR-binding function of NS5A, demonstrating that these properties may be linked to the IFN-resistant phenotype of HCV.     

Protein Kinase C-Activating Tumor Promoters Enhance the Differentiation of Astrocytes in Aggregating Fetal Brain Cell Cultures

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.     

Mutants of Salmonella typhimurium That Are Insensitive to Catabolite Repression of Proline Degradation

In Salmonella typhimurium the two enzymes of proline catabolism, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase, are subject to catabolite repression when the cells are grown in the presence of glucose. Mutants partially relieved of catabolite repression (PutR) for the proline catabolic enzymes have been isolated by selection on agar plates containing glucose and proline. The specificity of the catabolite repression-insensitive character for the enzymes of proline utilization has been confirmed by an analysis of other unrelated catabolic enzymes. Histidase and amylomaltase of the mutant strains are equally as sensitive to glucose repression as are the enzymes from the wild type. All four PutR mutants exhibit higher induced and higher basal levels of proline oxidase as compared with the corresponding wild-type levels. The mutations of three strains tested are cotransducible with constitutive, pleiotrophic-negative and structural gene mutations of the put region. Three-factor crosses indicate that two putR mutations are located at one end of the cluster of put mutations.     

Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62

Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879–12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.     

Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response

In vivo phosphorylation sites of the tobacco calcium-dependent protein kinases NtCDPK2 and NtCDPK3 were determined in response to biotic or abiotic stress. Stress-inducible phosphorylation was exclusively located in the variable N termini, where both kinases were phosphorylated differentially despite 91% overall sequence identity. In NtCDPK2, serine 40 and threonine 65 were phosphorylated within 2 min after stress. Whereas Thr⁶⁵ is subjected to intra-molecular in vivo autophosphorylation, Ser⁴⁰ represents a target for a regulatory upstream protein kinase, and correct NtCDPK2 membrane localization is required for Ser⁴⁰ phosphorylation. NtCDPK3 is phosphorylated at least at two sites in the N terminus by upstream kinase(s) upon stress stimulus, first at Ser⁵⁴, a site not present in NtCDPK2, and also at a second undetermined site not identical to Ser⁴⁰. Domain swap experiments established that differential phosphorylation of both kinases is exclusively determined by the respective N termini. A cell death-inducing response was only observed upon expression of a truncated variant lacking the junction and calcium-binding domain of NtCDPK2 (VK2). This response required protein kinase activity and was reduced when subcellular membrane localization was disturbed by a mutation in the myristoylation and palmitoylation site. Our data indicate that CDPKs are integrated in stress-dependent protein kinase signaling cascades, and regulation of CDPK function in response to in vivo stimulation is dependent on its membrane localization.     

KLHL31促进C2C12细胞的肌原分化

人类KLHL31基因是本实验室已克隆的基因,其蛋白质含有保守的BTB和串连重复的Kelch结构域,已有报道表明其在人类成体骨骼肌和心肌组织中特异表达。RT-PCR分析表明在C2C12细胞肌原分化过程中过表达KLHL31能够提高肌原分化标志基因MyoD与Myogenin的转录水平;荧光报告系统分析发现过表达KLHL31能够增强肌原分化相关基因MCK启动子的活性,表明KLHL31能够促进C2C12细胞的肌原分化。     

cAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress

Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.