Signaling to the DEAD box--regulation of DEAD-box p68 RNA helicase by protein phosphorylations

P68 nuclear RNA helicase is essential for normal cell growth. The protein plays a very important role in cell development and proliferation. However, the molecular mechanism by which the p68 functions in cell developmental program is not clear. We previously observed that bacterially expressed his-p68 was phosphorylated at multiple sites including serine/threonine and tyrosine [L. Yang, Z.R. Liu, Protein Expr. Purif., 35: 327]. Here we report that p68 RNA helicase is phosphorylated at tyrosine residue(s) in HeLa cells. Phosphorylation of p68 at threonine or tyrosine residues responds differently to tumor necrosis factor alpha (TNF-alpha)induced cell signal. Kinase inhibition and in vitro kinase assays demonstrate that p68 RNA helicase is a cellular target of p38 MAP kinase. Phosphorylation of p68 affects the ATPase and RNA unwinding activities of the protein. In addition, we demonstrate here that phosphorylation of p68 RNA helicase controls the function of the protein in the pre-mRNA splicing process. Interestingly, phosphorylation at different amino acid residues exhibits different regulatory effects. The data suggest that function(s) of p68 RNA helicase may be subjected to the regulation of multiple cell signal pathways.     

P68 RNA helicase mediates PDGF-induced epithelial mesenchymal transition by displacing Axin from beta-catenin

The nuclear p68 RNA helicase (referred to as p68) is a prototypical member of the DEAD box family of RNA helicases. The protein plays a very important role in early organ development. In the present study, we characterized the tyrosine phosphorylation of p68 under platelet-derived growth factor (PDGF) stimulation. We demonstrated that tyrosine phosphorylation of p68 at Y593 mediated PDGF-stimulated epithelial-mesenchymal transition (EMT). We showed that PDGF treatment led to phosphorylation of p68 at Y593 in the cell nucleus. The Y593-phosphorylated p68 (referred to as phosphor-p68) promotes beta-catenin nuclear translocation via a Wnt-independent pathway. The phosphor-p68 facilitates beta-catenin nuclear translocation by blocking phosphorylation of beta-catenin by GSK-3beta and displacing Axin from beta-catenin. The beta-catenin nuclear translocation and subsequent interaction with the LEF/TCF was required for the EMT process. These data demonstrated a novel mechanism of phosphor-p68 in mediating the growth factor-induced EMT and uncovered a new pathway to promote beta-catenin nuclear translocation.     

Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein

We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli. Huang et al. The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs. To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein. RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed. Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA. More interestingly, the C-terminal domain was a target of protein kinase C (PKC). Phosphorylation of the C-terminal domain of p68 abolished its RNA binding. Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68. The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein.     

Tyrosine phosphorylation of sam68 by breast tumor kinase regulates intranuclear localization and cell cycle progression

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44^mapk and p42^mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44^mapk and p42^mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues

We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the bacterially expressed p68 RNA helicase is phosphorylated on tyrosine, serine, and threonine residues. Our data demonstrated that phosphorylations on the recombinant p68 RNA helicase affect the enzymatic activities of the protein. This is the first observation that recombinant protein expressed in bacteria Escherichia coli is phosphorylated at multiple residues by bacterial endogenous protein kinases. Our observations suggest an important mechanism in controlling the function of p68 RNA helicase by signal transduction pathways.     

Anchorage-independent phosphorylation of p130(Cas) protects lung adenocarcinoma cells from anoikis

The regulation and function of the signaling adaptor protein p130(Cas) in tumor cell anchorage-independent survival, or anoikis resistance, were investigated in human lung adenocarcinoma cells. The tyrosine phosphorylation and function of p130(Cas) during cell detachment were analyzed in tumor cells and compared with that of normal epithelial cells. Cell detachment trigged rapid dephosphorylation of p130(Cas) in the nontumorigenic and anoikis-sensitive normal epithelial cells, but had no effect on the tyrosine phosphorylation of p130(Cas) in the anoikis-resistant lung adenocarcinoma cells. Further analysis revealed that the total tyrosine kinase activities associated with p130(Cas) in the lung tumor cells are anchorage-independent and are significantly higher than that in the normal cells, in which the p130(Cas)-associated tyrosine kinase activities are anchorage-dependent. Analysis of two known p130(Cas)-associated tyrosine kinases FAK and Src indicated that the regulation of tyrosine phosphorylation of FAK and Src are altered in the tumor cells. Inhibition of Src specifically abolished phosphorylation of p130(Cas) and induced anoikis. Furthermore, overexpression of dominant-negative forms of p130(Cas) also induced apoptosis. Taken together, these data suggest that p130(Cas) mediates a cell survival signal from cell-matrix interaction. Alterations in tumor cells that lead to constitutive phosphorylation of p130(Cas) can prevent cells from anoikis, hence contribute to tumor cell anchorage independence and metastasis.     

Hyperphosphorylated cortactin in cancer cells plays an inhibitory role in cell motility

Cortactin is frequently overexpressed in cancer cells, and changes of the levels of its tyrosine phosphorylation have been observed in several cancer cells. However, how the expression level and phosphorylation state of cortactin would influence the ultimate cellular function of cancer cells is unknown. In this study, we analyzed the role of cortactin in gastric and breast cancer cell lines using RNA interference technique and found that knockdown of cortactin inhibited cell migration in a subset of gastric cancer cells with a lower level of its tyrosine phosphorylation, whereas it greatly enhanced cell migration and increased tyrosine phosphorylation of p130Cas in other subsets of cells with hyperphosphorylated cortactin. Consistent results were obtained when hyperphosphorylation of cortactin was induced in MCF7 breast cancer cells by expressing Fyn tyrosine kinase. Additionally, immunostaining analysis showed that knockdown of hyperphosphorylated cortactin resulted in the recruitment of p130Cas to focal adhesions. These results suggest that cortactin hyperphosphorylation suppresses cell migration possibly through the inhibition of membrane localization and tyrosine phosphorylation of p130Cas.     

The ATPase, RNA unwinding, and RNA binding activities of recombinant p68 RNA helicase

p68 RNA helicase, a nuclear RNA helicase, was identified 2 decades ago. The protein plays very important roles in cell development and organ maturation. However, the biological functions and enzymology of p68 RNA helicase are not well characterized. We report the expression and purification of recombinant p68 RNA helicase in a bacterial system. The recombinant p68 is an ATP-dependent RNA helicase. ATPase assays demonstrated that double-stranded RNA (dsRNA) is much more effective than single-stranded RNA in stimulating ATP hydrolysis by the recombinant protein. Consistently, RNA-binding assays showed that p68 RNA helicase binds single-stranded RNA weakly in an ATP-dependent manner. On the other hand, the recombinant protein has very high affinity for dsRNA. Binding of the protein to dsRNA is ATP-independent. The data indicate that p68 may directly target dsRNA as its natural substrate. Interestingly, the recombinant p68 RNA helicase unwinds dsRNA in both 3' --> 5' and 5' --> 3' directions. This is the second example of a Asp-Glu-Ala-Asp (DEAD) box RNA helicase that unwinds RNA duplexes in a bi-directional manner.     

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src.

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation.

To elucidate the rapid events in signal transduction of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL 3), we examined phosphorylation of proteins on both serine and tyrosine residues in a cytokine-stimulated human myeloid cell line. We found increases in tyrosine phosphorylation within 30 s of stimulation with GM-CSF or IL 3, with peak responses occurring within 2 min. IL 3 and GM-CSF also induced serine phosphorylation, though 10 min of stimulation was required for maximum phosphate incorporation. Interestingly, both IL 3 and GM-CSF stimulated phosphate incorporation in identical substrates, a 68 kDa seryl-phosphoprotein (p68) and a 140 kDa tyrosyl-phosphoprotein (p140). Treatment of AML 193 cells with phorbol myristate acetate resulted in serine phosphorylation of p68; however, p140 was not phosphorylated on tyrosine. Depletion of protein kinase C isoenzymes with high concentrations of phorbol myristate acetate resulted in p68 phosphorylation, which was not further increased by IL 3 or GM-CSF. In contrast, cytokine-induced phosphorylation on tyrosine of p140 was observed after protein kinase C depletion. These data demonstrate the co-ordinate yet independent serine and tyrosine phosphorylation in IL 3- and GM-CSF-treated human myeloid cells, and thus suggest a common set of protein kinases stimulated by each separate ligand.     

Modulation of Janus kinase 2 by p53 in ovarian cancer cells

The constitutive activation of the Janus kinase 2 (JAK2) and mutation of the p53 tumor suppressor are both detected in human cancer. We examined the potential regulation of JAK2 phosphorylation by wild-type (wt) p53 in human ovarian cancer cell lines, Caov-3 and MDAH2774, which harbor mutant form of p53 tumor suppressor gene and high levels of phosphorylated JAK2. The wt p53 gene was re-introduced into the cells using an adenovirus vector. In addition to wt p53, mutant p53 22/23, mutant p53-175, and NCV (negative control virus) were introduced into the cells in the control groups. Expression of wt p53, but not that of p53-175 mutant, diminished JAK2 tyrosine phosphorylation in MDAH2774 and Caov-3 cell lines. Expression of wt p53 or p53 22/23 mutant did not cause a reduction in the phosphorylation of unrelated protein kinases, ERK1 and ERK2 (ERK1/2). The inhibition of JAK2 tyrosine phosphorylation can be reversed by tyrosine phosphatase inhibitor, sodium orthovanadate. Protein tyrosine phosphatase 1-B levels increased with introduction of wt p53 and may be involved in the dephosphorylation of JAK2. These findings present a possible p53-dependent cellular process of modulating JAK2 tyrosine phosphorylation in ovarian cancer cell lines.     

(-)-Epigallocatechin-3-gallate suppresses growth of AZ521 human gastric cancer cells by targeting the DEAD-box RNA helicase p68

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.     

Unwinding a path to nuclear beta-catenin

In this issue of Cell, Yang et al. (2006b) show that PDGF, a growth factor that induces the transition of epithelial cells to mesenchymal cells, stimulates the c-Abl kinase-dependent phosphorylation of p68 RNA helicase. Phosphorylated p68 dissociates beta-catenin from the Axin destruction complex, thereby promoting nuclear beta-catenin signaling independent of Wnt activation.     

A novel anti-DR5 chimeric antibody and epirubicin synergistically suppress tumor growth

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells. TRAIL receptor 2 (DR5) expression is high in tumor cells, transformed cells, and clinical tumor specimens and is low in most normal cells and tissues; therefore, DR5 is considered an attractive target for cancer therapy. In this study, HMCAZ5, a novel mouse-human chimeric antibody based on AD5-10, was generated and stably expressed in CHO-dhfr(-) cells. Highly purified HMCAZ5 exhibits a high affinity for the receptor that is equal to the parental mouse antibody, induces apoptosis in various cancer cells but not in normal hepatocytes, and elicits both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in various human cancer cells. The anthracycline anticancer drug epirubicin (EPB) synergizes the cytotoxicity of HMCAZ5 in cancer cells by upregulating DR5 expression on the cell surfaces, enhancing p53 expression, Bid cleavage, and JNK phosphorylation and downregulating c-FLIP expression and Akt phosphorylation. Moreover, HMCAZ5 alone suppresses tumor growth, and EPB augments the tumoricidal activity in human colorectal and hepatocellular tumor xenografts in athymic nude mice. These data suggest that the anti-DR5 chimeric antibody HMCAZ5 may have a clinical use and represents a useful immunological strategy, in combination with chemotherapy, for the treatment of cancer ? 2012 IUBMB Life, 64(9): 757-765, 2012.