Growth inhibition of human hepatocellular carcinoma cells by overexpression of G-protein-coupled receptor kinase 2

Hepatocellular carcinoma (HCC) is one of the deadliest forms of human liver cancer and does not respond well to conventional therapies. Novel effective treatments are urgently in need. G-protein-coupled kinase 2 (GRK2) is unique serine/threonine kinase that involves in many signaling pathways and regulates various essential cellular processes. Altered levels of GRK2 have been linked with several human diseases including cancer. In this study, we investigated a novel approach for HCC treatment by inducing overexpression of GRK2 in human HCC cells. We found that overexpression of GRK2 through recombinant adenovirus transduction inhibits the growth of human HCC cells. BrdU incorporation assay showed that the growth inhibition caused by elevated GRK2 level was due to reduced cell proliferation but not apoptosis. To examine the anti-proliferative function of increased GRK2 level, we performed cell cycle analysis using propidium iodide staining. We found that the proliferation suppression was associated with G2/M phase cell cycle arrest by the wild-type GRK2 but not its kinase-dead K220R mutant. Furthermore, increased levels of wild-type GRK2 induced upregulation of phosphor-Ser(15) p53 and cyclin B1 in a dose-dependent manner. Our data indicate that the anti-proliferative function of elevated GRK2 is associated with delayed cell cycle progression and is GRK2 kinase activity-dependent. Enforced expression of GRK2 in human HCC by molecular delivery may offer a potential therapeutic approach for the treatment of human liver cancer.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH₂ terminus, and a recent study showed that this NH₂-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.     

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.     

Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell proliferation and migration in cancer cells

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. Consistent with this function, tNOX knockdown by RNA interference leads to a significant reduction in cell proliferation and migration in HeLa cells, whereas tNOX overexpression confers an aggressive phenotype. Here, for the first time, we report that tNOX is phosphorylated by protein kinase Cδ (PKCδ) both in vitro and in vivo. Replacement of serine-504 with alanine significantly reduces phosphorylation by PKCδ. Co-immunoprecipitation experiments reveal an interaction between tNOX and PKCδ. Moreover, whereas overexpression of wild-type tNOX in NIH3T3 cells increases cell proliferation and migration, overexpression of the S504A tNOX mutant leads to diminished cell proliferation and migration, reflecting reduced stability of the unphosphorylatable tNOX mutant protein. Collectively, these results suggest that phosphorylation of serine-504 by PKCδ modulates the biological function of tNOX.     

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.     

RalGDS couples growth factor signaling to Akt activation

The Akt kinase is a key regulator of cell proliferation and survival. It is activated in part by PDK1-induced phosphorylation. Here we show that RalGDS, a Ras effector protein that activates Ral GTPases, has a second function that promotes Akt phosphorylation by PDK1 by bringing these two kinases together. In support of this conclusion is our finding that suppression of RalGDS expression in cells inhibits both epidermal growth factor and insulin-induced phosphorylation of Akt. Moreover, while PDK1 complexes with N-GDS, Akt complexes with the central region of RalGDS through an intermediary, JIP1. The biological significance of this newly discovered RalGDS function is highlighted by the observation that an N-terminally deleted mutant of RalGDS that retains the ability to activate Ral proteins but loses the ability to activate Akt also fails to promote cell proliferation. Thus, RalGDS forms a nexus that transduces growth factor signaling to both Ral GTPase and Akt-mediated signaling cascades.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.     

Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.     

Elevated Expression of the Serine-Arginine Protein Kinase 1 Gene in Ovarian Cancer and Its Role in Cisplatin Cytotoxicity In Vitro

Alternatively spliced variants of several oncogenes and tumor suppressors have been shown to be important for their tumorigenicity. In the present study we have tested whether serine-arginine protein kinase 1 (SRPK1), a major regulator of splicing factors, is involved in ovarian cancer progression and plays a role in chemo-sensitivity. By Western blot analyses, SRPK1 protein was found to be overexpressed in 4 out of 6 ovarian cancer cell lines as compared with an immortalized ovarian surface epithelial cell line; and in 55% of ovarian tumor samples as compared with non-neoplastic ovarian tissue samples. Reduction of SRPK1 expression using small interfering RNA (siRNA) encoding small hairpin RNA in ovarian cancer cells led to (i) reduced cell proliferation rate, slower cell cycle progression and compromised anchorage-independent growth and migration ability in vitro, (ii) decreased level of phosphorylation of multiple serine-arginine proteins, and P44/42MAPK and AKT proteins, and (iii) enhanced sensitivity to cisplatin. Together, these results suggest that elevated SRPK1 expression may play a role in ovarian tumorigenesis and SRPK1 may be a potential target for ovarian cancer therapy.     

The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.     

Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways

Lewis Y (LeY) is a carbohydrate tumor‐asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen‐activated protein kinases (MAPKs) and phosphoinositide‐3 kinase (PI3K)/Akt signaling pathways on FUT4‐induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4‐induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S‐phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin‐dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways. J. Cell. Physiol. 225: 612–619, 2010. © 2010 Wiley‐Liss, Inc.     

Mitogen- and stress-activated protein kinase 1 mediates activation of Akt by ultraviolet B irradiation

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.     

Reactive oxygen species regulate M-CSF-induced monocyte/macrophage proliferation through SHP1 oxidation

Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.     

CXCL12 induces tyrosine phosphorylation of cortactin, which plays a role in CXC chemokine receptor 4-mediated extracellular signal-regulated kinase activation and chemotaxis

CXC chemokine receptor 4 (CXCR4) plays a role in the development of immune and central nervous systems as well as in cancer growth and metastasis. CXCR4-initiated signaling cascades leading to cell proliferation and chemotaxis are critical for these functions. The present study demonstrated that stimulation of CXCR4 by its ligand, CXCL12, induced transient translocation of cortactin from endosomal compartments to the cell periphery where it colocalized with CXCR4 followed by internalization of CXCR4 together with cortactin into endosomes. Cortactin was co-immunoprecipitated with CXCR4 in response to CXCL12 treatment in a time-dependent manner. Ligand stimulation induced phosphorylation of cortactin at tyrosine 421, and the phosphorylation was both c-Src- and dynamin-dependent. Cortactin overexpression promoted CXCR4 internalization and recycling. However, overexpression of a cortactin mutant in which tyrosine 421 was replaced with alanine (cortactin-Y421A) or knockdown of cortactin with RNA interference (RNAi) reduced CXCR4 internalization in response to CXCL12. CXCR4-mediated activation of extracellular signal-regulated kinases 1 and 2 was significantly prolonged by overexpression of wild-type cortactin but not by the cortactin-Y421A mutant and was inhibited by cortactin knockdown with RNAi. Moreover, CXCL12-induced chemotaxis was enhanced by cortactin overexpression, reduced by overexpression of the cortactin-Y421A mutant, and blocked by cortactin knockdown with RNAi. These data provide strong evidence for an important role of cortactin in CXCR4 signaling and trafficking as well in the receptor-mediated cell migration.     

Functional analysis of α1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

The α1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of α1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe(x) were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.