Regulation Of Extracellular Signal-regulated Kinase Research Articles

Oxidant-Mediated cAMP Response Element Binding Protein Activation

Oxidant stress-mediated regulation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic outcomes in lung epithelium, yet a role for Ca2+ and Ca2+/cAMP-response element binding protein (CREB) in ERK1/2 signaling has not been defined. In this study, we tested the hypotheses that oxidants induce Ca2+-mediated phosphorylation of ERK and CREB, and that CREB is required for oxidant-induced proliferation and apoptosis. H2O2 initiated an influx of extracellular Ca2+ that was required for phosphorylation of both ERK and CREB in C10 lung epithelial cells. H2O2-mediated CREB phosphorylation was sensitive to MEK inhibition, suggesting that crosstalk between Ca2+, ERK, and CREB signaling pathways contributes to the oxidant-induced response. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos steady-state mRNA levels, but unexpectedly enhanced bcl-2 steady-state mRNA levels after H2O2 exposure. Whereas inhibition of CREB activity had no detectable effect on H2O2 stimulation of cell cycle, loss of CREB activity significantly reduced the number of cells undergoing apoptosis. These data support a novel communication between Ca2+-ERK1/2 and CREB elicited by H2O2, and further provide evidence that CREB is an important regulator of apoptosis in oxidant-mediated responses of lung epithelial cells.

Vanishin is a novel ubiquitinylated death-effector domain protein that blocks ERK activation

The ERK (extracellular-signal regulated-kinase)/MAPK (mitogen-activated protein kinase) pathway can regulate transcription, proliferation, migration and apoptosis. The small DED (death-effector domain) protein PEA-15 (phosphoprotein enriched in astrocytes-15) binds ERK and targets it to the cytoplasm. Other DED-containing proteins including cFLIP and DEDD can also regulate signal transduction events and transcription in addition to apoptosis. In the present study, we report the identification of a novel DED-containing protein called Vanishin. The amino acid sequence of Vanishin is closest in similarly to PEA-15 (61% identical). Vanishin mRNA is expressed in several mouse tissues and in both mouse and human cell lines. Interestingly, Vanishin is regulated by ubiquitinylation and subsequent degradation by the 26 S proteasome. The ubiquitinylation is complex and occurs at both the internal lysine residues and the N-terminus. We further show that Vanishin binds ERK/MAPK but not the DED proteins Fas-associated death domain, caspase 8 or PEA-15. Vanishin is present in both the nucleus and Golgi on overexpression and forces increased ERK accumulation in the nucleus in the absence of ERK stimulation. Moreover, Vanishin expression inhibits ERK activation and ERK-dependent transcription in cells, but does not alter MAPK/ERK activity. Therefore Vanishin is a novel regulator of ERK that is controlled by ubiquitinylation.

Extracellular Signal-regulated Kinases 1/2 Control Claudin-2 Expression in Madin-Darby Canine Kidney Strain I and II Cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.

Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus

In several neurological disorders including hyperhomocysteinemia, homocysteine (Hcy) accumulates in the brain, and acts as a potent neurotoxin. However, the molecular mechanisms induced by increased levels of Hcy in brain are not well understood. Here we show an activation of the extracellular signal-regulated kinases (ERK1 and ERK2) and the downstream nuclear targets Elk-1 and calcium/cAMP response element binding protein, in the hippocampus of cystathionine beta synthase deficient mice, a murine model of hyperhomocysteinemia. An ex vivo model of hippocampal slices allowed us to reproduce Hcy -induced ERK activation and to unravel the mechanisms responsible of this activation. Of interest, N-methyl- d-aspartate (NMDA), non-NMDA and metabotropic glutamate receptor antagonists all blocked Hcy -induced ERK activation. Moreover, the ERK activation was blocked in the presence of Na +-channel blocker tetrodotoxin, indicating the existence of a trans-synaptic activity in ERK activation by Hcy in hippocampal slices. The effects of Hcy on ERK cascade activation were also dependent on calcium influx, CaMK-II, PKC as well as PKA activation. Thus, altogether these data support a role of Hcy on ERK activation, via complex mechanisms, starting with a control of glutamate release, which in turn activates ionotropic and metabotropic receptor subtypes and produces increases in intracellular calcium levels.

Regulation of Ultraviolet B-induced Phosphorylation of Histone H3 at Serine 10 by Fyn Kinase

Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro, and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.

Depolarization Activates ERK and Proline-rich Tyrosine Kinase 2 (PYK2) Independently in Different Cellular Compartments in Hippocampal Slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.

PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells

Recent studies suggest that the tyrosine kinase Src plays an important role in the hormonal regulation of extracellular signal-regulated kinases (ERKs) via cyclic AMP (cAMP). Src has also been proposed to mediate signals downstream of nerve growth factor (NGF). Here, we report that the cAMP-dependent protein kinase A (PKA) induced the phosphorylation of Src at residue serine17 (S17) in multiple cell types including PC12, Hek293, AtT-20 and CHO cells. In PC12 cells, Src phosphorylation on S17 participates in the activation of the small G protein Rap1 by both cAMP and NGF. In these cells, Rap1 is required for cAMP/PKA signaling to ERKs and also for the sustained activation of ERKs by NGF. The activation of Rap1 by both cAMP and NGF was blocked by PP2, an inhibitor of Src family kinases, and by a Src mutant incapable of being phosphorylated by PKA (SrcS17A), consistent with the requirement of PKA phosphorylation of Src at S17 in these actions. PP2 and SrcS17A also inhibited the Rap1-dependent activation of ERKs by both agents. These results strongly indicate that PKA phosphorylation of Src at S17 is essential for cAMP and NGF signaling in PC12 cells and identify PKA as an important downstream target of NGF. PKA phosphorylation of Src may therefore be required for Rap1 activation in PC12 cells.

PKC-epsilon regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth.

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-epsilon isoform. To define the effectors of PKC-epsilon involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-epsilon (PKC-epsilon/WT) or dominant negative PKC-epsilon (PKC-epsilon/DN) in neonatal rat cardiocytes. PKC-epsilon/DN inhibited acute activation of PKC-epsilon produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-epsilon because PKC-epsilon/DN did not prevent translocation of either PKC-alpha or PKC-delta. Overexpression of PKC-epsilon/DN blunted the acute increase in ERK1/2 phorphorylation induced by the alpha(1)-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-delta with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-epsilon/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-epsilon/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-epsilon and that PE-induced growth is mediated by PKC-epsilon.

Leptin receptor signaling and the regulation of mammalian physiology.

While the hormone leptin and its receptor were discovered relatively recently, a great deal is already known about the molecular details of leptin receptor (LR) signaling and physiologic regulation. While multiple alternatively spliced LR isoforms exist, only the long (LRb) form associates with the Janus kinase 2 (Jak2) tyrosine kinase to mediate intracellular signaling. LRb initiates signaling via three major mechanisms: 1) Tyr(985) of LRb recruits SH2-containing tyrosine phosphatase (SHP-2); 2) Tyr(1138) of LRb recruits signal transducer and activator of transcription 3 (STAT3); and 3) tyrosine phosphorylation sites on the receptor-associated Jak2 likely recruit numerous undefined signaling proteins. The Tyr(985) --> SHP-2 pathway is a major regulator of extracellular signal-regulated kinase (ERK) activation during leptin signaling in cultured cells, while the Tyr(1138) --> STAT3 pathway induces the feedback inhibitor, suppressor of cytokine signaling 3 (SOCS3), as well as important positive effectors of leptin action. The Jak2-dependent activation of the insulin receptor substrate (IRS) protein --> phosphatidylinositol 3-kinase (PI3'-K) pathway appears to regulate membrane potential in LRb-expressing neurons and contributes to the regulation of feeding. The Tyr(1138) --> STAT3 pathway mediates transcriptional regulation of the hypothalamic melanocortin pathway in vivo. This pathway is required for the regulation of appetite and energy expenditure by leptin. Interestingly, the Tyr(1138) --> STAT3 pathway does not strongly regulate neuropeptide Y (NPY) and thus is not required for the control of reproduction and growth. Thus, other as-yet-undefined leptin receptor signals are central to these and perhaps other aspects of leptin action.

Degrading ERK3

Coulombe et al . discovered that, unlike other mitogen-activated protein kinases (MAPK), regulation of extracellular signal-regulated kinase 3 (ERK3) activity depended on the rate of protein degradation. The activity of MAPKs is primarily regulated through changes in subcellular localization and phosphorylation state. Coulombe et al . attempted to express ERK3 , a MAPK whose mRNA levels are regulated during development, in various cell types and discovered that, in contrast to other MAPKs, its protein was undetectable in cell lysates by Western analysis. The authors measured ERK3 stability by Western analysis of ERK3 in immunoprecipitates obtained at various times after pharmacological inhibition of protein synthesis and demonstrated that both endogenous and transfected ERK3 were highly unstable. Pharmacological analysis indicated that the ongoing degradation of ERK3 depended on ubiquitination and proteolysis by the 26S proteasome. Analysis of ERK1-ERK3 chimeric proteins identified two degradation regions in the ERK3 N-terminal lobe; insertion of this lobe into a stable protein confirmed that it functioned as a transferable degradation signal. In two models of cell differentiation (PC12 cells exposed to nerve growth factor and a line of myogenic cells grown in low serum), differentiation was accompanied by a rise in ERK3. In the myogenic cells, this increase in ERK3 largely depended on increased protein stability. In 3T3 fibroblasts, moreover, expression of a stabilized ERK3 mutant inhibited cell-cycle progression. These data suggest that, unlike other MAPKs, the activity of ERK3 is largely regulated through control of protein degradation. P. Coulombe, G. Rodier, S. Pelletier, J. Pellerin, S. Meloches, Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-associated protein kinase regulation during cellular differentiation. Mol. Cell. Biol. 23 , 4542-4558 (2003). [Abstract] [Full Text]

Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.

Regulation of Extracellular Signal-Regulated Kinase by Cannabinoids in Hippocampus

Endocannabinoids form a novel class of intercellular messengers, the functions of which include retrograde signaling in the brain and mediation or modulation of several types of synaptic plasticity. Yet, the signaling mechanisms and long-term effects of the stimulation of CB1 cannabinoid receptors (CB1-R) are poorly understood. We show that anandamide, 2-arachidonoyl-glycerol, and Delta9-tetrahydrocannabinol (THC) activated extracellular signal-regulated kinase (ERK) in hippocampal slices. In living mice, THC activated ERK in hippocampal neurons and induced its accumulation in the nuclei of pyramidal cells in CA1 and CA3. Both effects were attributable to stimulation of CB1-R and activation of MAP kinase/ERK kinase (MEK). In hippocampal slices, the stimulation of ERK was independent of phosphatidyl-inositol-3-kinase but was regulated by cAMP. The endocannabinoid-induced stimulation of ERK was lost in Fyn knock-out mice, in slices and in vivo, although it was insensitive to inhibitors of Src-family tyrosine kinases in vitro, suggesting a noncatalytic role of Fyn. Finally, the effects of cannabinoids on ERK activation were dependent on the activity of glutamate NMDA receptors in vivo, but not in hippocampal slices, indicating the existence of several pathways linking CB1-R to the ERK cascade. In vivo THC induced the expression of immediate-early genes products (c-Fos protein, Zif268, and BDNF mRNAs), and this induction was prevented by an inhibitor of MEK. The strong potential of cannabinoids for inducing long-term alterations in hippocampal neurons through the activation of the ERK pathway may be important for the physiological control of synaptic plasticity and for the general effects of THC in the context of drug abuse.

Negative Regulation of ERK and Elk by Protein Kinase B Modulates c-fos Transcription

In this study, we have identified novel regulatory steps involved in the cross-talk between protein kinase B (PKB) and MAPK signaling pathways. We found that PKB down-regulates the Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which leads to inactivation of the transcription factor Elk1. In addition, PKB is able to reduce protein levels of Elk1. Both events lead to suppression of serum response element (SRE)-dependent transcription and a consequent decrease in the transcription of SRE-containing genes, such as c-fos. Because activation of the Ras/MAPK cascade is reported to increase c-fos transcription before apoptosis, our results are consistent with a specific role for PKB in promoting cell survival. Decrease in c-Fos protein levels in glioblastoma cells with constitutively active PKB provides further support for our observations. Therefore, our findings delineate a novel mechanism regulating immediate-early transcription, which may be involved in the initial steps in PKB-induced oncogenic transformation.

Focus Issue: The Kinome--Techniques and Methods for Analysis

Focus Issue: The Kinome--Techniques and Methods for Analysis

A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK

cAMP is involved in a wide variety of cellular processes that were thought to be mediated by protein kinase A (PKA). However, cAMP also directly regulates Epac1 and Epac2, guanine nucleotide-exchange factors (GEFs) for the small GTPases Rap1 and Rap2 (refs 2,3). Unfortunately, there is an absence of tools to discriminate between PKA- and Epac-mediated effects. Therefore, through rational drug design we have developed a novel cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), which activates Epac, but not PKA, both in vitro and in vivo. Using this analogue, we tested the widespread model that Rap1 mediates cAMP-induced regulation of the extracellular signal-regulated kinase (ERK). However, both in cell lines in which cAMP inhibits growth-factor-induced ERK activation and in which cAMP activates ERK, 8CPT-2Me-cAMP did not affect ERK activity. Moreover, in cell lines in which cAMP activates ERK, inhibition of PKA and Ras, but not Rap1, abolished cAMP-mediated ERK activation. We conclude that cAMP-induced regulation of ERK and activation of Rap1 are independent processes.

Angiopoietin-1 and VEGF in vascular development and angiogenesis in hypoplastic lungs.

We hypothesized that exposure of murine fetuses to environmental toxins, such as nitrofen, during early embryogenesis alters vasculogenesis. To address our hypothesis, we assessed protein levels of endothelial cell-selective angiogenic factors: angiopoietin (ANG)-1, vascular endothelial growth factor (VEGF), and mediator of VEGF signaling, VEGF receptor-2 [fetal liver kinase (Flk)-1], a transmembrane receptor tyrosine kinase. VEGF and Flk-1 proteins were lower in hypoplastic lungs from pseudoglandular to alveolar stages than in normal lungs at equivalent developmental time points significant for induction of pulmonary vasculogenesis and angiogenesis. ANG-1 protein was higher in hypoplastic lungs than in normal lungs at all the developmental stages considered in this study, i.e., pseudoglandular, canalicular, saccular, and alveolar stages. We assessed exogenous VEGF-mediated endothelial cell response on extracellular signal-regulated kinase (ERK) 1/2, also referred to as p44/42 mitogen-activated protein kinase. Hypoplastic lungs had more elevated ERK 1/2 protein than normal developing lungs. Exposure to exogenous VEGF activated ERK 1/2 in normal developing lungs but not in hypoplastic lungs. Our results suggest that in hypoplastic lungs: 1) low VEGF signifies negative effects on vasculogenesis/angiogenesis and indicates altered endothelial-mesenchymal interactions; 2) increased ANG-1 protein may be required to maintain vessel integrity and quiescence; and 3) regulation of ERK 1/2 protein is affected in hypoplastic lungs. We speculate that extensive remodeling of blood vessels in hypoplastic lungs may occur to compensate for structurally and functionally defective vasculature.

Regulation of extracellular signal-regulated kinase cascades by alpha- and beta-isoforms of the human thromboxane A(2) receptor.

Thromboxane A(2) (TXA(2)) stimulates mitogenic growth of vascular smooth muscle. In humans, TXA(2) signals through two TXA(2) receptor (TP) isoforms, termed TPalpha and TPbeta. To investigate the mechanism of TXA(2)-mediated mitogenesis, regulation of extracellular signal-regulated kinase (ERK) signaling was examined in human embryonic kidney 293 cells stably overexpressing the individual TP isoforms. The TXA(2) mimetic 9,11-dideoxy-9alpha,11alpha-methano epoxy prostaglandin F(2alpha) (U46619) elicited concentration- and time-dependent activation of ERK1 and -2 through both TPs with maximal TPalpha- and TPbeta-mediated ERK activation observed after 10 and 5 min, respectively. U46619-mediated ERK activation was inhibited by the TP antagonist [1S-[1alpha,2beta-(5Z)-3beta,4alpha-]]-7-[3-[[2-(phenylamino)carbonyl]hydrazine] methyl]-7-oxabicyclo[-2,2,1-]hept-2yl]-5-heptenoic acid (SQ29,548), and by the mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Although ERK activation through TPalpha was dependent on 2-[1-(dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X)-sensitive protein kinase (PK) Cs, ERK activation through TPbeta was only partially dependent on PKCs. ERK activation through both TPalpha and TPbeta was dependent on PKA and phosphoinositide 3-kinase (PI3K) class 1(A), but not class 1(B), and was modulated by Harvey-Ras, A-Raf, c-Raf, and Rap1B/B-Raf and also involved transactivation of the epidermal growth factor receptor. Additionally, PKB/Akt was activated through TPalpha and TPbeta in a PI3K-dependent manner. In conclusion, we have defined the key components of TXA(2)-mediated ERK signaling and have established that both TPalpha and TPbeta are involved. TXA(2)-mediated ERK activation through the TPs is a complex event involving PKC-, PKA-, and PI3K-dependent mechanisms in addition to transactivation of the EGF receptor. TPalpha and TPbeta mediate ERK activation through similar mechanisms, although the time frame for maximal ERK activation and PKC dependence differs.

17 beta-estradiol induces spermatogonial proliferation through mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activity in the lizard (Podarcis s. sicula).

There are always more evidences indicating that 17beta-estradiol (E(2)) is necessary for normal male fertility. We have used a nonmammalian vertebrate model (the lizard Podarcis s. sicula) to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2) exerts a role in the spermatogenesis through ERK1/2 activity. Immunocytochemistry analysis shows that ERK1/2 proteins are present in the nucleus of the spermatogonia (SPG), and in primary (I) spermatocytes (SPC). The annual E(2) profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, ERK1/2 (molecular weight 44 and 42 kDa, respectively) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E(2) treatment induces spermatogonial proliferation, possibly via the activation of ERK1/2, and this effect is counteracted by the antiestrogen ICI 182-780.

Identification of dual specificity phosphatases induced by olfactory bulbectomy in rat olfactory neuroepithelium

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinases (MAPKs) which are target enzymes activated by a wide range of cell-surface stimuli. Like these kinases, a class of dsPTP has been implicated in cell differentiation, regeneration, and apoptosis. In order to isolate dsPTPs which might play an important role in neuronal regeneration and apoptosis in olfactory neuroepithelium, we subcloned DNA fragments amplified by reverse transcription-polymerase chain reaction (RT-PCR), using degenerate oligonucleotide primers based on the conserved amino acid regions within the catalytic domain of dsPTPs, from rat olfactory epithelial RNA 1 and 4 h after an olfactory bulbectomy. The PCR products were subcloned into the pCRII vector, and 23 clones were chosen for further characterization. The sequence of these 23 individual clones revealed that two clones were identical to the rat dsPTP, MKP-3, and the other 21 clones were identical to the rat dsPTP, MKP-1. By Northern analysis, the MKP-1 transcript was induced and peaked 4 h following a bulbectomy. Similar results were obtained with the MKP-3 transcript. These results suggest that MKP-1 and MKP-3 may be involved in the early steps of apoptosis in vivo in rat olfactory neuroepithelium.

Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell.

To examine the possible crosstalk and the roles of angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). Fetal VSMCs were prepared from rat fetal aorta at embryonic day 20. Expression of Ang II receptors was measured by a radioligand binding assay. Apoptotic changes were assessed by caspase 3 activity and chromatin dye staining. Regulation of extracellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determining phosphorylated ERK with Western blot. Ang II receptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by protein tyrosine phosphatase assay. The expression of AT1 and AT2 receptors was approximately 70%: 30% per cell. Serum depletion induced apoptosis in fetal VSMCs and selective AT1 receptor stimulation attenuated the apoptotic changes, whereas selective AT2 receptor activation enhanced apoptosis. Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of the AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Moreover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fetal VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a significant decrease of the AT2 receptor-mediated inhibition of ERK phosphorylation and attenuated the proapoptotic effect of AT2 receptor. These results indicate that a crosstalk between AT1 and AT2 receptors regulates the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling.