Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.     

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.     

Farrerol maintains the contractile phenotype of VSMCs via inactivating the extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase signaling

Molecular and Cellular Biochemistry - Farrerol, a dihydroflavone isolated from Rhododendron dauricum L., can inhibit vascular smooth muscle cell (VSMC) proliferation and exert a protective effect...     

Phosphatidylinositol 3-kinase-dependent extracellular calcium influx is essential for CX(3)CR1-mediated activation of the mitogen-activated protein kinase cascade.

Fractalkine, the first member of the CX(3)C chemokine family, induces leukocyte chemotaxis through activation of its high affinity receptor, CX(3)CR1. Like other chemokine receptors, CX(3)CR1 is coupled to a pertussis toxin-sensitive heterotrimeric G(i) protein, which is necessary for rapid rise in the concentration of intracellular calcium. Using a Chinese hamster ovary cell line stably transfected with the CX(3)CR1 receptor, we show that the source of calcium mobilized by fractalkine stimulation is the extracellular pool. Calcium influx is blocked by extracellular calcium chelators, as well as by divalent heavy metals such as Ni(2+), Co(2+), and Cd(2+) without affecting the integrity of intracellular stores. Remarkably, selective phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin and LY294002, abolish the wave extracellular calcium, suggesting that an active PI3K is necessary for this event. The influx of extracellular calcium is in turn required to trigger the activation of the p42/44 mitogen-activated protein/extracellular signal-regulated kinase pathway, but is not necessary for other signals downstream to PI3K, such as phosphorylation of Akt. The potential role of this signaling cascade in fractalkine-mediated chemotaxis is discussed.     

B cell receptor-mediated Syk-independent activation of phosphatidylinositol 3-kinase,Ras, and mitogen-activated protein kinase pathways

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.     

Nidus vespae protein inhibiting proliferation of HepG2 hepatoma cells through extracellular signal-regulated kinase signaling pathways and inducing G1 cell cycle arrest

A protein named NVP(1) was isolated from Nidus vespae. The aim of the present study was to elucidate whether and how NVP(1) modulates the proliferation of HepG2 cells. NVP(1) at a concentration of 6.6 microg/ml could arrest the cell cycle at stage G1 and inhibit the mRNA expression of cyclinB, cyclinD1 and cyclinE. NVP(1) suppressed cdk2 protein expression, but increased p27 and p21 protein expression. However, NVP(1) did not alter p16 protein expression levels. NVP(1) promoted apoptosis in HepG2 cells as indicated by nuclear chromatin condensation, and in addition, the extracellular signal-regulated kinase (ERK) signaling pathway was activated. Moreover, the p-ERK protein expression level was attenuated when the HepG2 cells were pretreated with ERK inhibitor PD98059. These results demonstrate that NVP(1) inhibits proliferation of HepG2 through ERK signaling pathway. NVP(1) could be a potential drug for liver cancer.     

Effects of resistance exercise intensity on extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation in men

Extracellular signal-regulated kinase (ERK) 1/2 signaling has been shown to be increased after heavy resistance exercise and suggested to play a role in the hypertrophic adaptations that are known to occur with training. However, the role that ERK1/2 may play in response to lower intensities of resistance exercise is unknown. Therefore, the purpose of this study was to determine the effects of resistance exercise intensity on ERK1/2 activity in human skeletal muscle. Twelve recreationally active men completed separate bouts of single-legged resistance exercise with 8-10 repetitions (reps) at 80-85% 1 repetition maximum (1RM) (85%) and 18-20 reps at 60-65% 1RM (65%) in a randomized crossover fashion. For both resistance exercise sessions, vastus lateralis biopsies and blood draws were taken immediately before exercise (PRE) and at 30 minutes (30MPST), 2 hours (2HRPST), and 6 hours (6HRPST) post exercise, with an additional blood draw occurring immediately after exercise (POST). The phosphorylated levels of pIGF-1R, pMEK1, pERK1/2, and activated Elk-1 were assessed by phosphoELISA, and serum insulin-like growth factor 1 (IGF-1) was assessed via enzyme-linked immunosorbent assay. Statistical analyses used a 2 × 4 (muscle responses) and 2 × 5 (serum responses) multivariate analysis of variance on delta values from baseline (p < 0.05). Both exercise intensities significantly increased the activity of insulin-like growth factor 1 receptor (IGF-1R), mitogen-activated protein kinase 1, ERK1/2, and Elk-1, with peak activity occurring at 2HRPST (p < 0.001). However, 65% resulted in a preferential increase in IGF-1R and Elk-1 activation when compared with 85% (p < 0.05). No differences were observed for serum IGF-1 levels regardless of intensity and time. These findings demonstrate that resistance exercise upregulates ERK1/2 signaling in a manner that does not appear to be preferentially dependent on exercise intensity.     

The anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate induces endothelial cell apoptosis by inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway

Imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is a new ruthenium compound active against lung metastasis in vivo and tumor cell invasion in vitro. Since angiogenesis was recognized as a key event in the metastasizing process, the manipulation of neo-vessel formation has been developed as a new therapeutic approach. Within this context, a pivotal role for apoptosis in regulating cellular growth has been proposed. In the present study, we exposed to NAMI-A the spontaneously transformed human endothelial cell line ECV304 and assessed a number of apoptosis-related features, including the DNA degradation rate, the activation of caspase-3 protease, the expression of Hsp27, and the release of cytochrome c. Cell treatment with NAMI-A elicited a significant increment in the apoptotic response, as indicated by DNA fragmentation and caspase-3 activation, two classical hallmarks of cellular suicide. Furthermore, NAMI-A was able to down-regulate Hsp27 protein expression and provoke the release of mitochondrial cytochrome c in the cytosol. Here, we analyze the involvement of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signal transduction pathway in the induction of apoptosis elicited by NAMI-A. Such a response was associated with a marked inhibition of MAPK/ERK kinase (MEK) and ERK phosphorylation with a time course and dose dependency overlapping those observed throughout NAMI-A-induced apoptosis. In addition, we report that PD98059, a selective MEK inhibitor, is able to induce apoptosis by itself in the ECV304 cell line. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating an apoptotic event in ECV304.     

The mechanism of dephosphorylation of extracellular signal-regulated kinase 2 by mitogen-activated protein kinase phosphatase 3.

The mitogen-activated protein (MAP) kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERKs). Inactivation of MAP kinases occurs by dephosphorylation of Thr(P) and Tyr(P) in the TXY kinase activation motif. To gain insight into the mechanism of ERK2 inactivation by MKP3, we have carried out an analysis of the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2. We find that ERK2/pTpY dephosphorylation by MKP3 involves an ordered, distributive mechanism in which MKP3 binds the bisphosphorylated ERK2/pTpY, dephosphorylates Tyr(P) first, dissociates and releases the monophosphorylated ERK2/pT, which is then subjected to dephosphorylation by a second MKP3, yielding the fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a k(cat)/K(m) of 3.8 x 10(6) m(-1) s(-1), which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide encompassing the pTEpY motif. This strikingly high substrate specificity displayed by MKP3 may result from a combination of high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and specific ERK2-induced allosteric activation of the MKP3 C-terminal phosphatase domain.     

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.     

Mitogen-activated protein kinase/extracellular signal-regulated kinase attenuates 3-hydroxykynurenine-induced neuronal cell death

3-Hydroxykynurenine (3-HK), an endogenous tryptophan metabolite, is known to have toxic effects in brain. However, the molecular mechanism of the toxicity has not been well identified. In this study, we investigated the involvement of MAPK/extracellular signal-regulated kinase (ERK) in the 3-HK-induced neuronal cell damage. Our results showed that 3-HK induced apoptotic neuronal cell death and ERK phosphorylation occurred during cell death. Inhibition of ERK activation using PD98059 considerably increased cell death. Furthermore, cell death was preceded by mitochondrial malfunction including collapse of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to the cytosol. Interestingly, inhibition of ERK dramatically increased mitochondrial malfunction, and enhanced caspase activation, resulting in enhanced neuronal cell death. Thus, our results show that ERK plays a protective role by maintaining mitochondrial function and regulating caspase activity under conditions of cellular stress.     

Glycogen synthase kinase 3 beta is a natural activator of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1)

Glycogen synthase kinase 3beta (GSK3 beta) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and insulin response. GSK3 beta has now been shown to induce activation of the mitogen-activated protein kinase kinase kinase MEKK1 and thereby to promote signaling by the stress-activated protein kinase pathway. GSK3 beta-binding protein blocked the activation of MEKK1 by GSK3 beta in human embryonic kidney 293 (HEK293) cells. Furthermore, co-immunoprecipitation analysis revealed a physical association between endogenous GSK3 beta and MEKK1 in HEK293 cells. Overexpression of axin1, a GSK3 beta-regulated scaffolding protein, did not affect the physical interaction between GSK3 beta and MEKK1 in transfected HEK293 cells. Exposure of cells to insulin inhibited the activation of MEKK1 by GSK3 beta, and this inhibitory effect of insulin was abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Furthermore, MEKK1 activity under either basal or UV- or tumor necrosis factor alpha-stimulated conditions was reduced in embryonic fibroblasts derived from GSK3 beta knockout mice compared with that in such cells from wild-type mice. Ectopic expression of GSK3 beta increased both basal and tumor necrosis factor alpha-stimulated activities of MEKK1 in GSK3 beta(-/-) cells. Together, these observations suggest that GSK3 beta functions as a natural activator of MEKK1.     

Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.     

The extracellular signal-regulated kinase mitogen-activated protein kinase/ribosomal S6 protein kinase 1 cascade phosphorylates cAMP response element-binding protein to induce MUC5B gene expression via D-prostanoid receptor signaling

Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D(2,) an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD(2) binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD(2) induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD(2) expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD(2) increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD(2)-induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD(2) induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD(2)-induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD(2) caused an increase in intracellular cAMP levels, whereas intracellular Ca(2+) did not have such an effect. PGD(2)-induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (-921/-914 and -900/-893). Finally, we demonstrated that PGD(2) can induce MUC5B overproduction via ERK MAPK/RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway.     

Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.     

Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn(-/-) mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn(-/-) mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.     

Keratins modulate c-Flip/extracellular signal-regulated kinase 1 and 2 antiapoptotic signaling in simple epithelial cells

Among the large family of intermediate filament proteins, the keratin 8 and 18 (K8/K18) pair constitutes a hallmark for all simple epithelial cells, such as hepatocytes and mammary cells. Functional studies with different cell models have suggested that K8/K18 are involved in simple epithelial cell resistance to several forms of stress that may lead to cell death. We have reported recently that K8/K18-deprived hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis. Here we show that upon Fas, tumor necrosis factor alpha receptor, or tumor necrosis factor alpha-related apoptosis-inducing ligand receptor stimulation, an inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation sensitizes wild-type but not K8-null mouse hepatocytes to apoptosis and that a much weaker ERK1/2 activation occurs in K8-null hepatocytes. In turn, this impaired ERK1/2 activation in K8-null hepatocytes is associated with a drastic reduction in c-Flip protein, an event that also holds in a K8-null mouse mammary cell line. c-Flip, along with Raf-1, is part of a K8/K18-immunoisolated complex from wild-type hepatocytes, and Fas stimulation leads to further c-Flip and Raf-1 recruitment in the complex. This points to a new regulatory role of simple epithelium keratins in the c-Flip/ERK1/2 antiapoptotic signaling pathway.     

Nuclear localization of phospholipase D1 mediates the activation of nuclear protein kinase C(alpha) and extracellular signal-regulated kinase signaling pathways

Recent studies highlight the existence of a nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PC as a source of second messengers and, particularly, nuclear localization of PC-specific phospholipase D (PLD). In the present study we have identified the nuclear localization sequence (NLS) of PLD1 whose mutation abolished its nuclear import. Recently, we reported that caspase-mediated cleavage of PLD1 generates the N-terminal fragment (NF-PLD1) and C-terminal fragment (CF-PLD1). Here we show that CF-PLD1 but not NF-PLD1, is exclusively imported into the nucleus via its functional NLS, whereas only some portions of intact PLD1 were localized into the nucleus. The NLS of intact PLD1 or CF-PLD1 is required for interaction with importin-β, which is known to mediate nuclear import. The amount of intact PLD1 or CF-PLD1 translocated into nucleus is correlated with its binding affinity with importin-β. Ultimately, nuclear localization of intact PLD1 but not CF-PLD1 mediates the activation of nuclear protein kinase Cα and extracellular signal-regulated kinase signaling pathways. Taken together, we propose that nuclear localization of PLD1 via the NLS and its interaction with importin-β may provide new insights on the functional role of nuclear PLD1 signaling.     

Isolation of the protein kinase TAO2 and identification of its mitogen-activated protein kinase/extracellular signal-regulated kinase kinase binding domain.

We previously reported the cloning of the thousand and one-amino acid protein kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase sterile 20 protein. Here we report the complete sequence and properties of a related rat protein kinase TAO2. Like TAO1, recombinant TAO2 selectively activated mitogen-activated protein/extracellular signal-regulated kinase kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells. To examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was localized to an approximately 135-residue sequence just C-terminal to the TAO2 catalytic domain. In vitro this MEK binding domain associated with MEKs 3 and 6 but not MEKs 1, 2, or 4. Using chimeric MEK proteins, we found that the MEK N terminus was sufficient for binding to TAO2. Catalytic activity of full-length TAO2 enhanced its binding to MEKs. However, neither the autophosphorylation of the MEK binding domain of TAO2 nor the activity of MEK itself was required for MEK binding. These results suggest that TAO proteins lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets.     

Ligand binding up-regulates EphA2 messenger RNA through the mitogen-activated protein/extracellular signal-regulated kinase pathway

The EphA2 receptor tyrosine kinase is overexpressed in aggressive cancer cells, where it critically influences many aspects of malignant character. Although high levels of EphA2 have been documented in many different cancers, relatively little is known of the mechanisms that govern EphA2 gene expression in normal or malignant cells. Our present studies demonstrate that EphA2 influences the regulation of its own gene expression. Specifically, ligand-mediated phosphorylation of EphA2 transmits signals to the nucleus via extracellular signal-regulated kinase kinases to up-regulate de novo EphA2 gene expression and synthesis. This mechanism governs EphA2 expression in normal and malignant cells. In normal cells, EphA2 protein expression is balanced by ligand-mediated induction of EphA2 gene expression countered by EphA2 protein turnover. These findings suggest that EphA2 expression and ligand binding are intimately linked in epithelial cells. Increased understanding of this mechanism could have important implications for understanding the causes of EphA2 overexpression and for developing new strategies for therapeutic intervention in the many cancers that overexpress EphA2.