FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.     

Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.     

Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK

DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein–protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.     

Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.     

Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.     

Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation

TP53-induced glycolysis and apoptosis regulator (TIGAR) knockdown is proven to radiosensitize glioma cells, but the mechanisms are not fully understood. Thioredoxin-1 (TRX1) is a redox-sensitive oxidoreductase, which plays critical roles in DNA damage signal transduction via nuclear translocation in irradiated cells. Because the TRX1-dependent DNA damage signaling pathway relies on NADPH to maintain the reduced state of TRX1, and TIGAR functions to increase NADPH generation under oxidative stress, in this study, the role of TRX1 in TIGAR abrogation-induced radiosensitization was investigated. It was demonstrated that ionizing radiation (IR)-induced nuclear translocation of TRX1 was significantly inhibited by TIGAR interference and reversed by wild-type (WT)-TRX1 overexpression. In addition, WT-TRX1 overexpression could accelerate the process of DNA damage repair postponed by TIGAR knockdown in irradiated glioma cells. The reduction process of IR-oxidized TRX1 was also delayed by TIGAR knockdown but restored by WT-TRX1 overexpression. Therefore, we conclude that TIGAR knockdown-induced radiosensitization of glioma cells may be dependent on the inhibition of TRX1 nuclear translocation.     

Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle

Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.     

Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments

Actin depolymerizing factor (ADF)/cofilin enhances turnover of actin filaments by severing and depolymerizing filaments. A number of proteins functionally interact with ADF/cofilin to modulate the dynamics of actin filaments. Actin-interacting protein 1 (AIP1) has emerged as a conserved WD-repeat protein that specifically enhances ADF/cofilin-induced actin dynamics. Interaction of AIP1 with actin was originally characterized by a yeast two-hybrid system. However, biochemical studies revealed its unique activity on ADF/cofilin-bound actin filaments. AIP1 alone has negligible effects on actin filament dynamics, whereas in the presence of ADF/cofilin, AIP1 enhances filament fragmentation by capping ends of severed filaments. Studies in model organisms demonstrated that AIP1 genetically interacts with ADF/cofilin and participates in several actin-dependent cellular events. The crystal structure of AIP1 revealed its unique structure with two seven-bladed beta-propeller domains. Thus, AIP1 is a new class of actin regulatory proteins that selectively enhances ADF/cofilin-dependent actin filament dynamics.     

Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.     

Granulocyte colony-stimulating factor induces ERK5 activation, which is differentially regulated by protein-tyrosine kinases and protein kinase C. Regulation of cell proliferation and survival

Granulocyte colony-stimulating factor (G-CSF) plays a major role in the regulation of granulopoiesis. Treatment of cells with G-CSF has been shown to activate multiple signal transduction pathways. We show here that Erk5, a novel member of the MAPK family, and its specific upstream activator MEK5 were activated in response to incubation of cells with G-CSF. Different from other members of the MAPK family including Erk1/2, JNK, and p38, maximal activation of Erk5 by G-CSF required the C-terminal region of the G-CSF receptor. Genistein, a specific inhibitor of protein-tyrosine kinases, blocked G-CSF-induced Erk5 activation. In contrast, inhibition of protein kinase C activity increased G-CSF-mediated activation of Erk5 and MEK5, whereas stimulation of protein kinase C activity inhibited activation of the two kinases by G-CSF. The proliferation of BAF3 cells in response to G-CSF was inhibited by expression of a dominant-negative MEK5 but potentiated by expression of a constitutively active MEK5. Expression of the constitutively active MEK5 also increased the survival of BAF3 cells cultured in the absence of or in low concentrations of G-CSF. Together, these data implicate Erk5 as an important signaling component in the biological actions of G-CSF.     

B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK

The protein phosphatase 2A (PP2A) acts on several kinases in the extracellular signal-regulated kinase (ERK) signaling pathway but whether a specific holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown. By using both RNA interference and overexpression of PP2A B regulatory subunits, we show that B56, but not B, family members of PP2A increase ERK dephosphorylation, without affecting its activation by MEK. Induction of the early gene product and ERK substrate IEX-1 (ier3) by growth factors leads to opposite effects and reverses B56-PP2A-mediated ERK dephosphorylation. IEX-1 binds to B56 subunits and pERK independently, enhances B56 phosphorylation by ERK at a conserved Ser/Pro site in this complex and triggers dissociation from the catalytic subunit. This is the first demonstration of the involvement of B56-containing PP2A in ERK dephosphorylation and of a B56-specific cellular protein inhibitor regulating its activity in an ERK-dependent fashion. In addition, our results raise a new paradigm in ERK signaling in which ERK associated to a substrate can transphosphorylate nearby proteins.     

Novel nuclear translocation of inositol polyphosphate 4-phosphatase is associated with cell cycle,proliferation and survival

Inositol polyphosphate 4 phosphatase type I enzyme (INPP4A) has a well-documented function in the cytoplasm where it terminates the phosphatidylinositol 3-kinase (PI 3-K) pathway by acting as a negative regulator. In this study, we demonstrate for the first time that INPP4A shuttles between the cytoplasm and the nucleus. Nuclear INPP4A is enzymatically active and in dynamic equilibrium between the nucleus and cytoplasm depending on the cell cycle stage, with highest amounts detected in the nucleus during the G₀/G₁ phase. Moreover, nuclear INPP4A is found to have direct proliferation suppressive activity. Cells constitutively overexpressing nuclear INPP4A exhibit massive apoptosis. In human tissues as well as cell lines, lower nuclear localization of INPP4A correlate with cancerous growth. Together, our findings suggest that nuclear compartmentalization of INPP4A may be a mechanism to regulate cell cycle progression, proliferation and apoptosis. Our results imply a role for nuclear-localized INPP4A in tumor suppression in humans.     

Akt down-regulates ERK1/2 nuclear localization and angiotensin II-induced cell proliferation through PEA-15

Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.     

Cyclophilin A is required for CXCR4-mediated nuclear export of heterogeneous nuclear ribonucleoprotein A2, activation and nuclear translocation of ERK1/2, and chemotactic cell migration

The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.     

Response to mechanical strain in an immortalized pre-osteoblast cell is dependent on ERK1/2

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.     

Mitogen-activated protein kinase phosphatase PHS1 is retained in the cytoplasm by nuclear extrusion signal-dependent and independent mechanisms

The organization of plant microtubule arrays is thought to be regulated by phosphorylation and other signaling cascades, but the molecular components involved are largely unknown. We have previously found that a dominant missense mutation (phs1-1) in a putative kinase-docking motif of an Arabidopsis PHS1 phosphatase, which belongs to the mitogen-activated protein kinase phosphatase (MKP) family, compromises the stability of cortical microtubules. We here report that suppressor screening of phs1-1 recovered several intragenic recessive mutations in PHS1. In contrast to our previous report, null alleles of PHS1 were almost indistinguishable from the wild type in morphology, but their roots skewed to the abnormal direction when grew in the presence of low doses of a microtubule-destabilizing drug. PHS1 is mainly expressed in elongating cells, where the protein was distributed in the cytoplasm, predominantly in a microsomal fraction. Recruitment of green fluorescent protein-tagged PHS1 in endomembrane aggregates after treatment with brefeldin A or in an endomembrane-organization mutant suggests that an association with endomembranes retains PHS1 in the cytoplasm. A nuclear export signal identified in the C-terminal tail also contributes to the robust cytoplasmic retention of PHS1.