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To examine estrogen-induced growth mechanisms of endometrial carcinoma, we investigated the estrogen-induced activation of the mitogen-activated protein kinase (MAPK) pathway and cell cycle regulators. Estradiol (E2) treatment at concentrations of 10−8 M and 10−6 M to estrogen receptor (ER)-positive endometrial carcinoma Ishikawa cells for 24 h resulted in increased cell proliferation by 20% and 28% respectively. The E2-induced proliferation was associated with the activation of extracellular signal-regulated kinase (MAPK)3/1 and up-regulation of cyclin D1 and E, which were suppressed by the addition of an MAP2K inhibitor (U0126) or an ER antagonist (ICI 182 780). Then, our screening for estrogen-inducible growth factors identified that IGF1 was up-regulated remarkably by E2. Immunoprecipitation using conditioned medium of Ishikawa cells after E2 treatment confirmed the E2-induced secretion of IGF1 protein. Treatment with recombinant IGF1 stimulated cell proliferation in a dose-dependent fashion, in association with MAPK3/1 phosphorylation and up-regulation of cyclin D1 and E. These IGF1-induced responses were suppressed by treatment with MAP2K inhibitor or anti-IGF1 receptor antibody. Immunohistochemical staining confirmed the expression of activated MAPK3/1 in normal proliferative phase endometria and endometrial carcinomas, indicating the involvement of this pathway in actively proliferating endometrial tissues in vivo. These findings suggest that E2-induced proliferation of endometrial carcinoma cells is mediated by the MAPK3/1 pathway via autocrine stimulation of IGF1.

MAP kinase-activated protein kinase 5 (MK5) was originally described as a protein kinase activated downstream of the p38 MAP kinase and is also named p38-regulated/activated protein kinase (PRAK). However, while MK5 is most similar in sequence to the two p38 regulated MAPKAP kinases MK2 and MK3, recent data has shown that in contrast to these enzymes MK5 is not activated in response to either cellular stress or pro-inflammatory cytokines. This lack of response to stimuli which cause robust activation of p38 MAP kinase in vivo is supported by data obtained using transgenic mice lacking MK5. Unlike animals lacking MK2 and MK3, MK5 null mice respond normally to endotoxic shock and display an unchanged pattern of cytokine expression in response to LPS. Clues as to the physiological function of MK5 have come from the recent observation that MK5 is uniquely regulated and activated following complex formation with the atypical MAP kinases ERK3 and ERK4. Thus, it is possible that MK5 is unique amongst the MAPKAP kinases in being regulated downstream of signaling pathways other than the classical MAP kinases p38 and ERK1/2.

Background Luminal pressure may injure the gut mucosa in obstruction, ileus, or inflammatory bowel disease. Methods We formed Roux-en-Y anastomoses in 19 mice, creating proximal and defunctionalized partially obstructed limbs and a distal limb to vary luminal pressure and flow. We induced mucosal ulcers by serosal acetic acid, and assessed proliferation (proliferating cell nuclear antigen) and ERK (immunoblotting). Parallel studies compared Caco-2 enterocyte migration and proliferation after pressure and/or ERK blockade. Results At 3 days, anastomoses were probe-patent, proximal and distal limbs contained chyme, and defunctionalized limbs were empty. The proximal and defunctionalized limbs showed increased pressure and slower healing despite increased proliferation, ERK protein, and ERK activation. In vitro, pressure decreased Caco-2 migration across collagen or fibronectin, stimulated proliferation, and activated ERK. However, ERK blockade did not prevent pressure effects. Conclusions Luminal pressure during obstruction or ileus may impair mucosal healing independently of luminal flow despite increased mitosis and ERK activation.

Apelin signaling to the family of mitogen-activated protein kinases (MAPKs), such as extracellular-regulated kinases 1/2 (ERK1/2) and p38 MAPK, through the coupling of apelin receptor (APJ) to G-protein, mediates important pathophysiological responses. Although apelin fragments have been reported to induce ERK1/2 activation through Gi-protein, the intracellular pathways by which APJ activates these MAPKs are only partially understood. Here, using stably transfected human embryonic kidney 293 (HEK293) cells overexpressing human APJ (HEK293-apelinR), we showed that apelin-13 signaling leads to ERK1/2 and p38 MAPK pathways through APJ activation. It was found in HEK293-apelinR cells that ERK1/2 activation was initiated by apelin-13 at 5 min, with the peak of activation occurring at 15 min, and a return to the basal level within 60 min. The activation of ERK1/2 appeared to be dose-dependent with a significant activation being observed at 10 nM apelin-13 and maximal activation at 100 nM. However, phosphorylated-p38 MAPK was not detected in HEK293-apelinR cells treated with apelin-13. We also shown that the apelin-13-induced ERK1/2 activation requires a coupling with pertussis toxin-sensitive G-protein, and that overexpression of dominant-negative Gi2 completely inhibits the apelin-13-induced ERK1/2 activation. In addition, treatment with apelin-13 resulted in a concentration-dependent reduction of forskolin-stimulated cAMP production. It is therefore suggested that apelin-13 activates ERK1/2 but not p38 MAPK, which involves the coupling of APJ to the Gi2 cascade. In conclusion, the ERK1/2, but not p38 MAPK pathway is activated by apelin-13 through coupling of human APJ to Gi2-protein, which contributes to cellular responses.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of plant growth, development and responses to a wide variety of stimuli. In a conditional gain-of-function transgenic system, the activation of AtMEK5, a MAPK kinase, can in turn activate endogenous AtMAPK3 and AtMAPK6, and can lead to a striking increase in ethylene production and induce hypersensitive response (HR)-like cell death in Arabidopsis. However, the role of the increased ethylene production in regulating this HR-like cell death remains unknown. Using Arabidopsis transgenic plants that express AtMEK5(DD), an active mutant of AtMEK5 that is under the control of a steroid-inducible promoter, we tested the contribution of ethylene to cell death. We found that ethylene biosynthesis occurs before cell death. Cell death was delayed by inhibiting AtMEK5-induced ethylene production using inhibitors of ACC-synthases, ACC-oxidases or ethylene receptors. In the mutants AtMEK5(DD)/etr1-1 and AtMEK5(DD)/ein2-1, both of which showed insensitivity to ethylene, the expression of AtMEK5(DD) protein, activity of AtMAPK3 and AtMAPK6, and ethylene production were the same as those seen in AtMEK5(DD) transgenic plants, but cell death was also delayed. These data suggest that ethylene signaling perception is required to accelerate cell death that is induced by AtMEK5 activation.

Cdc14 is a member of the dual-specificity phosphatase family, which is essential for faithful cell cycle progression in eukaryotic cells of different origin. The function of human Cdc14A (hCdc14A), however, has not been fully elucidated as only few physiological substrates have been identified. To gain insight into the biological role of Cdc14A, we performed a yeast two-hybrid screen designed to isolate substrates of this human phosphatase. Using this genetic approach, we here report the identification of Erk3, an atypical mitogen-activated protein kinase (MAPK), as a specific binding partner of hCdc14A. GST pull-down assays show that Erk3 interacts directly with hCdc14A in vitro via its unique C-terminal domain. Furthermore, biochemical analysis reveals that hCdc14A can remove cyclin-dependent kinase (Cdk)-mediated phosphorylation of Erk3 in vitro raising the possibility that Erk3 may be a potential substrate for hCdc14A in vivo. Consistent with a physiologically relevant cross-talk in vivo, we find that Cdc14A forms a stable complex with Erk3 in human cells independent of its intrinsic phosphatase activity but mediated by its regulatory C-terminal domain. We show that hCdc14A impacts the emerging signaling pathway between Erk3 and MK5, a MAPK-activated protein kinase. We document that hCdc14A upregulation leads to redistribution of the Erk3 substrate MK5 from the nucleus to the cytoplasm. In addition, we find that hCdc14A stabilizes complex formation between Erk3 and its binding partner cyclin D3, a D-type cyclin implicated in both cellular proliferation and differentiation. Collectively, our findings suggest an intimate functional relationship between the Cdc14A phosphatase and the Erk3 kinase in signaling pathways that regulate key cell-fate decisions in human cells.

Backgrounds Fibroblast growth factor 8-isoform b (FGF8b) has been detected in human clinical sex-organ related cancers including hormone-refractory prostate cancer. There are, however, few relevant experimental models. A murine monoclonal anti-FGF8 antibody, KM1334, has been shown to neutralize FGF8b and inhibit the growth of androgen-dependent mouse mammary SC-3 cells in vitro and in vivo. In the present study, we evaluated the anti-tumor activity of KM1334 against androgen-dependent and -independent progression of FGF8b-expressing human prostate cancer xenografts. Methods FGF8b cDNA was transfected into androgen-dependent human prostate cancer cell line LNCaP, and its xenograft tumors were established subcutaneously in SCID mice with or without castration. KM1334 at the dose of 400 µg/head was injected twice weekly. Results FGF8b-expressing LNCaP cells secreted FGF8b, showed enhanced level of Erk1/2 phosphorylation, and showed more potent growth properties than mock-expressing cells in vitro and in vivo. KM1334 reduced these properties in vitro, inhibited tumorigenecity in vivo (T/C = 0.33), and showed anti-tumor activity against established tumors (T/C = 0.47) of FGF8b-expressing cells. FGF8b-expressing LNCaP tumors were androgen-dependent. However, they recurred as androgen-independent FGF8b positive tumors after castration. KM1334 also inhibited the growth of established FGF8b-expressing tumors in the androgen-independent states (T/C = 0.47). Conclusions These results indicate that humanized monoclonal antibodies, conserving the paratope of KM1334, are a promising candidate for therapy of FGF8b-expressing clinical prostate cancers. Follow-up studies using xenograft models with clinical FGF8b-expressing tumors are required to validate these early findings. Prostate 68: 640–650, 2008. © 2008 Wiley-Liss, Inc.

OBJECTIVE—Protein kinase C (PKC) θ activation is associated with insulin resistance and obesity, but the underlying mechanisms have not been fully elucidated. Impairment of insulin-mediated vasoreactivity in muscle contributes to insulin resistance, but it is unknown whether PKCθ is involved. In this study, we investigated whether PKCθ activation impairs insulin-mediated vasoreactivity and insulin signaling in muscle resistance arteries. RESEARCH DESIGN AND METHODS—Vasoreactivity of isolated resistance arteries of mouse gracilis muscles to insulin (0.02–20 nmol/l) was studied in a pressure myograph with or without PKCθ activation by palmitic acid (PA) (100 μmol/l). RESULTS—In the absence of PKCθ activation, insulin did not alter arterial diameter, which was caused by a balance of nitric oxide–dependent vasodilator and endothelin-dependent vasoconstrictor effects. Using three-dimensional microscopy and Western blotting of muscle resistance arteries, we found that PKCθ is abundantly expressed in endothelium of muscle resistance arteries of both mice and humans and is activated by pathophysiological levels of PA, as indicated by phosphorylation at Thr538 in mouse resistance arteries. In the presence of PA, insulin induced vasoconstriction (21 ± 6% at 2 nmol/l insulin), which was abolished by pharmacological or genetic inactivation of PKCθ. Analysis of intracellular signaling in muscle resistance arteries showed that PKCθ activation reduced insulin-mediated Akt phosphorylation (Ser473) and increased extracellular signal–related kinase (ERK) 1/2 phosphorylation. Inhibition of PKCθ restored insulin-mediated vasoreactivity and insulin-mediated activation of Akt and ERK1/2 in the presence of PA. CONCLUSIONS—PKCθ activation induces insulin-mediated vasoconstriction by inhibition of Akt and stimulation of ERK1/2 in muscle resistance arteries. This provides a new mechanism linking PKCθ activation to insulin resistance.

The extracellular-regulated kinase (ERK) 4 (MAPK4) and ERK3 (MAPK6) are structurally related atypical MAPKs displaying major differences only in the C-terminal extension. ERK3 is known as an unstable mostly cytoplasmic protein that binds, translocates, and activates the MAPK-activated protein kinase (MK) 5. Here we have investigated the stability and expression of ERK4 and have analyzed its ability to bind, translocate, and activate MK5. We show that, in contrast to ERK3, ERK4 is a stable protein that binds to endogenous MK5. Interaction of ERK4 with MK5 leads to translocation of MK5 to the cytoplasm and to its activation by phosphorylation. In transfected HEK293 cells, where overexpressed catalytically dead ERK3 is able to activate MK5, catalytic activity of ERK4 is necessary for activation of MK5, indicating that ERK4 directly phosphorylates MK5. Interestingly, ERK4 dimerizes and/or oligomerizes with ERK3, suggesting that overexpressed inactive ERK3 recruits active endogenous ERK4 to MK5 for its activation. Hence, ERK3 and ERK4 cooperate in activation of MK5.

Several forms of cancer are characterized by frequent activating mutations in the serine/threonine kinase, BRAF. Substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of all BRAF activating mutations and leads to stimulation of kinase activity, downstream signaling, and cell transformation. To better understand the molecular pathogenesis induced by oncogenic BRAF signaling, we used microarray gene expression profiling to comprehensively analyze the BRAF-directed transcriptional program of cells expressing a conditionally active form of BRAFV600E. Several novel genes that affect proliferation, cell survival, angiogenesis and immune surveillance were identified as possible mediators of BRAF-induced oncogenic signaling. Moreover, we show that a MAPK family member, extracellular signal-regulated kinase-3 (ERK3/MAPK6) is highly expressed in response to BRAF signaling in this system. Cellular ERK3 protein is highly unstable and pharmacological inhibition of BRAF activity resulted in rapid ERK3 degradation. In melanoma cells, RNAi-mediated knockdown of endogenous BRAF or treatment with MEK inhibitors that prevent ERK1/2 activation led to a reduction in ERK3 levels, indicating that elevated ERK3 expression is mediated through MEK1/2 signaling. These results provide strong evidence for another mode by which BRAF can regulate the ERK protein kinase family and suggest ERK3 to be a potential pharmacodynamic marker for targeting BRAF signaling in melanoma.

Background. The aim of this study was to investigate the effects of adenovirus-mediated antisense ERK2 (Adanti-ERK2) gene therapy on chronic allograft nephropathy. Methods. We employed a rat kidney transplantation mode (F344→Lewis) and studied four groups: (1) controls (n = 6); (2) vector controls (n = 6); (3) an Adanti-ERK2 group (n = 10); and (4) an isograft group (n = 4). The animals were monitored for proteinuria, graft histology, infiltrating cells, and immune-related gene (interleukin-2 [IL-2] and intracellular adhesion molecule-1 [ICAM-1]) expression for 20 weeks after transplantation. Results. The control group had increasing proteinuria during the 20-week follow-up. All rats showed advanced chronic renal failure associated with strong immune cell infiltration and immune gene expression. Chronic graft injury was accelerated in the vector-control group, but no significant difference was observed compared with the control group. In contrast, the Adanti-ERK2 group showed less inflammation and improved graft histology/ function compared with controls. Moreover, ERK2 protein expression in the Adanti-ERK2 group was lower than in the control group (P

The adaptation of pancreatic islets to pregnancy includes increased beta cell proliferation, expansion of islet mass, and increased insulin synthesis and secretion. Most of these adaptations are induced by prolactin (PRL). We have previously described that in vitro PRL treatment increases ERK3 expression in isolated rat pancreatic islets. This study shows that ERK3 is also upregulated during pregnancy. Islets from pregnant rats treated with antisense oligonucleotide targeted to the PRL receptor displayed a significant reduction in ERK3 expression. Immunohistochemical double-staining showed that ERK3 expression is restricted to pancreatic beta cells. Transfection with antisense oligonucleotide targeted to ERK3 abolished the insulin secretion stimulated by glucose in rat islets and by PMA in RINm5F cells. Therefore, we examined the participation of ERK3 in the activation of a cellular target involved in secretory events, the microtubule associated protein MAP2. PMA induced ERK3 phosphorylation that was companied by an increase in ERK3/MAP2 association and MAP2 phosphorylation. These observations provide evidence that ERK3 is involved in the regulation of stimulus-secretion coupling in pancreatic beta cells. ispartof: Molecular and Cellular Endocrinology vol:251 issue:1-2 pages:33-41 ispartof: location:Ireland status: published

Mitogen-activated protein kinase (MAPK) cascades are activated in plants during responses to pathogens or to pathogen-derived elicitors and mediate intracellular stress responses. Here, we show that a rice (Oryza sativa) MAPK, OsMAPK6, was posttranslationally activated in a cell culture by a sphingolipid elicitor. Suppression of OsMAPK6 expression by RNA interference resulted in a strong reduction of pathogen-induced Phe ammonia-lyase mRNA, whereas the mRNA level of another rice MAPK, OsMAPK5a, was highly increased. Silencing of a small GTPase, OsRac1, by RNA interference or loss-of-function mutation (d1) of the heterotrimeric G-protein alpha-subunit gene resulted in a strong reduction of the OsMAPK6 protein levels and of kinase activation by a sphingolipid elicitor. Furthermore, coimmunoprecipitation experiments with OsRac1 and OsMAPK6 proteins showed that OsMAPK6 is closely associated with the active form of OsRac1, but not with inactive forms of OsRac1. These results indicate that these two G-proteins regulate an elicitor-inducible MAPK in rice at the protein level.

Fibroblast Growth Factor-2 (FGF-2) induces cell proliferation, cell migration, embryonic development, cell differentiation, angiogenesis and malignant transformation. The four forms of FGF-2 (Low Molecular Weight) and (High Molecular Weights) are alternative translation products, and have a different subcellular localization: the high molecular weight (HMWFGF-2) forms are nuclear while the low molecular weight form, (LMWFGF-2) is mainly cytoplasmic. Our previous work demonstrated NIH 3T3 cells expressing different FGF-2 forms, displayed a different phenotype, suggesting that nuclear and cytoplasmic forms of FGF-2 may have different functions. Here we report a cDNA microarray-based study in NIH 3T3 fibroblasts expressing different FGF-2 forms. Several candidate genes that affect cell-cycle, tumor suppression, adhesion and transcription were identified as possible mediators of the HMWFGF-2 phenotype and signaling pattern. These results demonstrated that HMWFGF-2 and LMWFGF-2 target the expression of different genes. Particularly, our data suggest that HMWFGF-2 forms may function as inducers of growth inhibition and tumor suppression activities.

To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.Western blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3, p38 and mitogen or ERK activated protein kinaseMEK-1 proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients. Immunohistochemistry was employed for their localization.Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526+/-65 760 vs 122 807+/-65 515, P = 0.001), ERK-2 (168 471+/-95 051 vs 120 469+/-72 874, P0.001), ERK-3 (118 651+/-71 513 vs 70 934+/-68 058, P0.001), P38 (104 776+/-51 650 vs 82 930+/-40 392, P = 0.048) and MEK-1 (116 486+/-45 725 vs 101 434+/-49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging (average ratio of integral optic density (IOD)(tumor): IOD(normal) in TNM I, II, III, IV tumors was 1.43+/-0.34, 5.08+/-3.74, 4.99+/-1.08, 1.44+/-1.02, n = 42, P = 0.023) and serosa invasion (4.31+/-4.34 vs 2.00+/-2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage III and IV tumors were higher than those in stage I and II tumors (2.64+/-3.01 vs 1.01+/-0.33, P = 0.022; 2.05+/-1.54 vs 1.24+/-0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49+/-2.91 vs 1.03+/-0.36, P = 0.023; 1.98+/-1.49 vs 1.24+/-0.44, P = 0.036) or serosa invasion (2.39+/-2.82 vs 1.01+/-0.35, P = 0.022; 1.95+/-1.44 vs 1.14+/-0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann II tumors, expression of ERK-2 and ERK-3 was increased compared with Borrmann III tumors (2.57+/-1.86 vs 1.23+/-0.60, P = 0.022; 5.50+/-5.05 vs 1.83+/-1.21, P = 0.014). Borrmann IV tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P0.05). Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site.MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers. Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

International audience; The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism

We have isolated a mouse cDNA for a novel dual-specificity phosphatase designated LDP-3 (low-molecular-mass dual-specificity phosphatase 3). The 450 bp open reading frame encodes a protein of 150 amino acids with a predicted molecular mass of 16 kDa. Northern blot and reverse transcription–PCR analyses show that LDP-3 transcripts are expressed in almost all mouse tissues examined. In vitro analyses using several substrates and inhibitors indicate that LDP-3 possesses intrinsic dual-specificity phosphatase activity. When expressed in mammalian cells, LDP-3 protein is localized mainly to the apical submembrane area. Forced expression of LDP-3 does not alter activation of ERK (extracellular-signal-regulated kinase), but rather enhances activation of JNK (c-Jun N-terminal kinase) and p38 and their respective upstream kinases MKK4 (mitogen-activated protein kinase kinase 4) and MKK6 in cells treated with 0.4 M sorbitol. By screening with a variety of stimuli, we found that LDP-3 specifically enhances the osmotic stress-induced activation of JNK and p38.

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

The ability of cancer cells to proliferate abnormally and invade surrounding tissue is among the most important features of the malignant phenotype. The mechanisms by which the mitogen activated protein kinase (MAPK) cascade regulates these phenotypes have been investigated for many years. Activated GTP bound Ras binds the upstream protein kinases Raf-1 and B-Raf. The MAPK kinases 1 and 2, known as MKK or MEK, are phosphorylated and activated by Raf. MEKs phosphorylate the extracellular signal regulated kinases ERK1 and ERK2. A unique member of the MAPK family is p97MAPK, the human homolog of rat ERK3. p97MAPK is ubiquitously expressed but whether its cellular functions are different from ERK1 and ERK2 is unknown. p97MAPK is highly expressed in human cancer cell lines. In the present study, expression of p97MAPK was unique among the ERK family in that its expression was induced when human carcinoma cell lines are plated on type IV collagen. This increased expression correlated with slower cancer cell proliferation on collagen compared to plastic tissue culture dishes. Overexpression of p97MAPK was sufficient to inhibit cellular proliferation with concomitant changes in cell cycle regulatory protein expression. p97MAPK also inhibited cancer cell migration and invasion by decreasing Rac1 expression but not that of matrix metalloproteinase 9 which is regulated by other ERKs. These data represent the first reported function of p97MAPK in human cancer cells.

Alterations in the intracellular signal transduction pathway in primary afferents may contribute to pain hypersensitivity. We demonstrated that very rapid phosphorylation of p38 mitogen-activated protein kinase occurred in dorsal root ganglion (DRG) neurons that were participating in the transmission of noxious signals. Capsaicin injection induced phosphorylated-p38 (p-p38) in small-to-medium diameter sensory neurons with a peak at 2 min after capsaicin injection. Furthermore, we examined the p-p38 labeling in the DRG after noxious thermal stimuli and found a stimulus intensity-dependent increase in labeled cell size and the number of activated neurons. Most of these p-p38-immunoreactive (IR) neurons were small- and medium-sized neurons, which coexpressed transient receptor potential ion channel TRPV1 and phosphorylated-extracellular signal-regulated protein kinase. Intrathecal administration of the p38 inhibitor, FR167653, reversed the thermal hyperalgesia produced by the capsaicin injection. Inhibition of p38 activation was confirmed by the decrease in the number of p-p38-IR neurons in the DRG following capsaicin injection. Taken together, these findings suggest that the activation of p38 pathways in primary afferents by noxious stimulation in vivo may be, at least in part, correlated with functional activity, and further, involved in the development of thermal hyperalgesia.

The mitogen activated serine/threonine kinases (MAPKs) constitute extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPK, with an important role in cell proliferation and transformation. Earlier studies from our laboratory had indicated a role for MAPK pathway in oral cancer. Our current study was aimed at examining the role of a MAPK-ERK3, in chewing-tobacco associated oral squamous cell carcinoma. We constructed a cDNA library from primary oral cancer tissue, cloned and isolated the ERK3 gene. The gene was sequenced and the sequence submitted to GenBank (Accession number AF420474). The oral cancer ERK3 clone demonstrated 100% homology to human ERK3 isolated from fetal skeletal muscle, with four specific nucleotide alterations in the non-coding region of the gene, comprising deletion of 'TTT' between 2701 and 2705 nt; 'G' to 'T' substitution at 188 nt; insertion of 'A' between 121 and 122 nt, and insertion of 'CTTTA' between 3391 and 3392 nt. Southern analysis of EcoRI genomic digests indicated ERK3 specific fragments of 11, 8.6, 6.5 and 3.2 kb sizes. The mRNA transcript analysis defined a single transcript of 4.5 kb. RT-PCR analysis revealed a three- to eight-fold increase in ERK3 expression in a majority (90%) of oral cancer tissues and peripheral blood cells (61.5%) of the patients, whereas absence or low levels of expression was observed in peripheral blood cells of 74% clinically normal healthy individuals with no tobacco habits, and overexpression in PBC from 26% normal individuals. The alterations in the non-coding region of ERK3 gene cloned from oral cancer tissue, may affect stability or regulation of mRNA, resulting in overexpression in the patient samples. The overexpression of the gene in the normal healthy individuals may be indicative of increased risk of developing oral cancers in this group.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogen-activated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.

Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G(1) arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.

The ability to express and purify proteins in large amounts through recombinant DNA technology has enabled significant advances in the biomedical sciences. Several commercially available or home-made vectors allow expression and easy purification of recombinant proteins in Escherichia coli. Generally, such vectors are designed to produce fusion proteins containing a molecular tag at the N-terminal end. Calmodulin, streptavidin, maltose binding protein, thioredoxin, hexahistidine ðHis6Þ, and glutathione S-transferase (GST) are examples of tags chosen for their high binding affinity toward specific ligands [7]. However, this procedure also has inherent limitations. In some cases, proteins overexpressed in E. coli form insoluble aggregates that are difficult to solubilize or are found mainly as truncated forms [1]. Purification of recombinant fusion proteins sometimes requires several chromatography steps and renaturation protocols. These problems are particularly evident for large proteins, for which yields of purification are frequently very low and preclude further analysis or use of the recombinant protein. Although several procedures have been developed to improve protein solubility in E. coli [1,2,6,8], less progress has been made to overcome the problem of truncated protein expression due to proteolysis, intrinsic instability, or premature translation termination caused by codon usage bias [4]. Here we describe the construction of novel dual-tag prokaryotic expression vectors that allow for enrichment in full-length recombinant proteins and facilitate their subsequent purification by sequential chromatography on metal and glutathione affinity columns. Extracellular signal-regulated kinase 3 (ERK3) is a member of the MAP kinase family of serine/threonine kinases [5]. In the course of our studies on this enzyme, we experienced problems in producing good yields of full-length GST fusion constructs of ERK3 using conventional prokaryotic expression systems. To circumvent these problems, we created a novel set of dual-tag prokaryotic expression vectors, pHGST.1 and pHGST. 2T, that were engineered to produce N-terminal His6and C-terminal GST-tagged fusion proteins (Fig. 1). In theory, the sequential purification of recombinant proteins on glutathione and metal affinity resins should: (1) allow for enrichment in full-length protein and (2) facilitate the purification process and increase the purity of the final product. pHGST vectors were constructed by first subcloning the two annealed oligonucleotides 50 CTA GCA TGA ATT CGG GAT CCA TGG GTC GAC TCG AGC TCG GAA 30 and 50 AGC TTT CCG AGC TCG AGT CGA CCC ATG GAT CCC GAA TTC ATG 30 into the NheI/HindIII sites of pRSET-A (Invitrogen Life Technologies, Burlington, Canada) to generate pRSET-MCS. The GST coding sequence was amplified by PCR using pGEX-KG [3] as template with the following oligonucleotides: 50 GCC GAG CTC TCC CCT ATA CTA GGT TAT TGG 30 and 50 CCC AAG CTT TTA ATC CGA TTT TGG AGG ATG GTC 30. The amplicon was then digested with SstI/ HindIII and subcloned into the SstI/HindIII sites of pRSET-MCS to yield pHGST.1. The pHGST.2T vector was obtained by ligation of the following annealed oligonucleotides into NcoI/XhoI-digested pHGST.1: 50 CAT GGC GGC CGC CTC GAG TCT GGT TCC Analytical Biochemistry 310 (2002) 219–222

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to induce apoptosis in various types of tumors, including prostate cancer. We sought to examine the key mechanisms affecting the resistance to 4-HPR-induced apoptosis in three human prostate cancer cell lines, PC-3, DU145, and LNCaP. Concentrations of more than 40 microM 4-HPR produced apoptosis to almost the same extent in all cell lines; however, only the LNCaP line remained highly sensitive to concentrations less than 10 microM. These differing sensitivities at low concentrations correlated well with the level of constitutive activation of nuclear factor kappa B (NFkappaB) in the individual cell lines. We found that NFkappaB activation inhibited c-jun NH(2)-terminal kinase and caspase 3 activation induced by 4-HPR and that NFkappaB inhibition by the I kappa B alpha phosphorylation inhibitor compound Bay 117082 resulted in increasing sensitization of both PC-3 and DU145 lines to apoptosis induced by 4-HPR at low concentrations. Furthermore, we found that inhibition of extracellular signal-regulated kinase (ERK) enhanced the suppression of NFkappaB by 4-HPR and also resulted in sensitization to apoptosis in the DU145 cell line, in which ERK is activated constitutively. It thus appears that mitogen-activated protein kinase associated with the activity of NFkappaB plays an important role in the degree of resistance to 4-HPR-induced apoptosis in human prostate cancer cells.

Objective: The intracellular pathway and the regulation of inducible nitric oxide synthase (iNOS) expression in skeletal muscle is incompletely understood. In vitro studies, using different cell types, suggest that inflammatory cytokines are potential triggers to induce iNOS expression. Methods: To analyze intracellular pathways leading to iNOS induction, rat skeletal myoblasts were incubated with inflammatory cytokines and assessed for iNOS expression by Western blot and Griess reaction. To confirm the in vitro findings, local cytokine levels were determined in skeletal muscle biopsies of patients with chronic heart failure (CHF) and correlated with iNOS expression. Results: Nitrite accumulation in the myoblast culture supernatant or iNOS protein in the cell pellet was significantly increased after incubation with IL-1β in combination with γ-IFN. Priming experiments revealed that γ-IFN elevated the expression of IL-1β receptor mRNA, whereby IL-1β was able to induce iNOS expression. The cytokine-mediated iNOS induction was significantly reduced by blocking ERK1/ERK2 activation and completely abolished by the inhibition of NFκB. In skeletal muscle biopsies of CHF patients the local content of IL-1β was significantly increased as compared to healthy controls. Furthermore, a linear correlation between IL-1β content and iNOS expression in the skeletal muscle was detected. Conclusions: These data demonstrate that IL-1β, together with the priming effect of γ-IFN, induces iNOS expression in skeletal muscle via activation of ERK1/ERK2 and NFκB.

The activation of natural killer (NK) cells and induction of cytotoxicity are complex processes whose molecular mechanisms have not been clearly elucidated. Stimulation of the NKL human NK cell line with interleukin-2 (IL-2) or protein-bound polysaccharide K (PSK) leads to sustained growth and cytolytic activity in comparison to unstimulated NKL cells. Our previous results shown that IL-2 and PSK regulate different nuclear transcription factors in NKL cells, and that the signal transduction pathway used by these inducers is different. To determine the molecular basis for the different action of IL-2 and PSK, we investigated the upstream effects generated in human NKL cells by IL-2 and PSK on protein kinase C (PKC) isoenzymes and mitogen-activated protein kinases (MAPK). Here we report the profile of unstimulated NKL cells as: PKCbetaPKCalphaPKCdelta =PKCepsilon. The PKCeta form was not expressed. The effects of PSK and IL-2 on these isoenzymes were different. IL-2 increased the expression of PKCalpha, PKCdelta and PKCepsilon, whereas PSK decreased the expression of PKCalpha, and also increased PKCdelta and PKCepsilon to higher levels than did IL-2. In MAPK expression we found that unstimulated NKL cells have the following profile: ERK2ERK6p38gammap38betaERK1. ERK3, ERK3 rel, ERK5/ERK4 and p38delta were not expressed. IL-2 decreased the expression of ERK2, whereas PSK did not, and both agents increased the expression of ERK3. These results shown that PSK and IL-2 produce different variations in PKC isoenzymes and MAPK in NKL cells.

Emerging evidence showed miR499a could not only function as an oncogene but also as a tumor suppressor in various types of cancer, such as melanoma. However, whether miR499a was involved in hepatocarcinogenesis remains unknown. We previously reported that miR499a was up-regulated in HBV-mediated hepatocellular carcinoma (HCC). In this study, we found that HBV could induce the expression of miR499a by promoting its promoter activity. In addition, we reported that miR499a increased cell proliferation and cell migration of HCC cells. MAPK6 was further identified as a target of miR499a, which could also be down-regulated by HBV. Moreover, we demonstrated that MAPK6 could rescue the cell growth induced by miR499a and HBV. These findings indicated that miR499a might play an oncogene role by targeting MAPK6 in the development and progression of HBV-related HCC.

Activation of the mitogen-activated protein kinases (MAPKs) appears to play an important role in both proliferation and transformation of various cells; the role of MAPK activation in colorectal cancers has not been clearly defined. The purpose of our study was to determine whether MAPK activity and protein levels were increased in colorectal cancers.Colorectal cancers and adjacent normal mucosa from 21 patients were extracted for protein. Expression levels and activity of the MAPKs (ERK1/2, JNK1, p38 and ERK3) were assessed by immunoblot analysis and in vitro kinase assays, respectively. In addition, changes in myelin basic protein (MBP) kinase activity and autophosphorylation were determined by in-gel kinase assays.The activities of ERK1/2, JNK1 and p38 were downregulated in the majority of cancers; ERK3 kinase activity was increased in 10 of 21 cancers. The presence of proteins displaying increased MBP phosphorylation and autophosphorylation was identified specifically in the cancers by in-gel kinase assays.Our findings demonstrate that the constitutive activation of ERK1/2, JNK1 and p38 is not a feature of colorectal cancers. Moreover, our in-gel kinase results suggest that protein kinases, other than the MAPKs assessed, may play a more crucial role in colon carcinogenesis.

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

In mammalian B lymphocytes, engagement of the B cell antigen receptor (BCR) activates several parallel intracellular signaling pathways which ultimately lead to expression of differentiated functions such as cell proliferation and antibody production or to cellular apoptosis. BCR engagement stimulates the classical mitogen activated protein kinase (MAPK) pathway, also called the extracellular-related kinase (ERK) pathway, resulting in activation of the signature terminal enzyme in the pathway, MAPK (or ERK). BCR signaling also activates the phosphatidyl inositol pathway and its key enzyme protein kinase C (PKC). To investigate the ERK pathway in cells of the teleost immune system, peripheral blood leukocytes from red drum or channel catfish were treated with PKC activators or antibodies which crosslink the BCR. Proteins were identified in both red drum and catfish B cells that resembled mammalian ERKs in molecular weight and in their possessing a distinctive pTEpY dual phosphorylation site. BCR-mediated activation of these presumptive teleost ERKs depended in part (red drum) or in total (catfish) on PKC. To our knowledge this represents the first report of a functional MAPK kinase pathway in teleost fish.

It is now well established that neurotrophins play a crucial role in the development of the nervous system. However, there is increasing evidence that the function of neurotrophins persists throughout adulthood. The broad scope of neurotrophin action is well documented in the case of nerve growth factor (NGF) and its effect on nociceptors and nociception. Here, we review the evidence for these multiple roles for NGF. Two manipulations influencing NGF levels are discussed in detail. The first involves the use of transgenic mice that overexpress or underexpress neurotrophins. A second strategy involves administration of NGF or its antibody in vivo to increase or decrease its level. During prenatal development, NGF is required for survival of nociceptors. In the early postnatal period, NGF is required for expression of the appropriate nociceptor phenotype. In adults, NGF acts as an important intermediate in inflammatory pain, contributing to both peripheral and central sensitization. The sensitization of peripheral nociceptors can be very rapid and can involve non-neural cells such as mast cells, neutrophils, fibroblasts, and macrophages. Recent evidence indicates that other neurotrophins also play key supporting roles in the development of nociceptors (e.g., NT-3) and in inflammatory pain (e.g., BDNF, NT-4/5). Furthermore, molecules from other superfamilies (e.g., GDNF) also are required to assure survival of certain classes of nociceptors. The diverse effects of neurotrophins on nociceptive processing emphasize their broad importance in the development and function of the nervous system.

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser189, comparable to Thr183, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.

In a prior study, we have shown that stable transfection of expression plasmids for protein kinases C beta 1 (PKC beta 1) or PKC beta 2 into differentiated colon cancer cells led to elevated levels of PKC beta 1 or PKC beta 2 protein and PKC beta kinase activities in the transfectants, without altering PKC alpha levels. PKC gamma is not found in these cells, so the major modulation was in PKC beta. PKC beta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKC beta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKC beta 1 and each of two PKC beta 2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKC beta 1, PKC beta 2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a10-fold increase in ERK3 activity in each PKC beta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKC beta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKC beta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKC beta 1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKC beta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKC beta 1 or PKC beta 2.

The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp) expected to be involved in catalysis eliminated autophosphorylation. Ser of ERK3, which corresponds to Thr, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions.

The biological activity of nerve growth factor (NGF) has been shown to be mediated by the p140trkA receptor tyrosine kinase, while the role of the p75 NGF receptor (p75NGFR) is still unresolved. Here we have investigated the relative contribution of p140trkA and p75NGFR to early consequences of NGF binding: ligand internalization, p140trkA autophosphorylation, and tyrosine phosphorylation of Shc, phospholipase Cγ-1 (PLCγ-1), and extracellular signal-regulated kinases (ERKs). It was found that NGF internalization was neither prevented by blocking p140trkA activity using the protein kinase inhibitors methylthioadenosine, staurosporine, and K-252a, nor by inhibiting NGF binding to p75NGFR with antibodies. However, when NGF binding to p140trkA was reduced by the use of a synthetic peptide corresponding to amino acids 36–53 of human p140trkA, internalization of NGF was decreased. Thus, at least in PC12 cells, internalization appears to require binding of NGF to p140trkA, but occurs irrespective of p140trkA kinase activity and ligand occupancy of p75NGFR. The NGF triple mutant Lys-32/Lys-34/Glu-35 to Ala, which has been demonstrated to bind to p140trkA, but not to p75NGFR, induced tyrosine phosphorylation more rapidly than wild-type NGF. Likewise, NGF-induced tyrosine phosphorylation was accelerated when NGF binding to p75NGFR was prevented with REX-IgG. These findings indicate that NGF binding by p75NGFR may modulate NGF-induced p140trkA kinase activity. © 1994 Wiley-Liss, Inc.

The activator protein-1 (AP-1) transcription factor modulates expression of genes involved in growth regulation, differentiation, and neoplastic transformation. Several mitogen-activated protein kinases (MAP kinases) as well as other kinases phosphorylate c-Jun and c-Fos in vitro and are postulated to control AP-1 activity. However, since many protein kinases phosphorylate substrates in vitro with which they have no association in vivo, we sought evidence for interaction in vivo between AP-1 and MAP kinase proteins. We now report detection of an association in vivo of MAP kinase-related proteins with c-Jun and AP-1 dimers by peptide mapping and two-dimensional electrophoretic analyses of proteins co-immunoprecipitated with AP-1 antigens. Extracellular signal-regulated kinase-2 and several apparently novel MAP kinase-related proteins are among the species that bind to AP-1. The large number of MAP kinase-related proteins associated with AP-1 implicates them on an important gene regulation pathway. Combinatorial association between MAP kinase-related proteins and AP-1 dimers could potentially create numerous distinct complexes that could regulate diverse genes.

Mitogen-activated protein (MAP) kinases [also known as Erks] have been established to function as important mediators of signal transduction by growth factor receptors. Several components of the MAP kinase signal transduction pathway have been demonstrated to be oncogenically activated in malignant tumors. These include growth factor receptors, the GTP-binding protein Ras, and the protein kinase Raf. The genes that encode MAP kinases therefore represent potential targets of carcinogenic insults. Here, we report the genomic loci of three MAP kinase genes are widely distributed within the human genome: p41mapk (Erk2) at 22q11.2; p44mapk (Erk1) at 16p11.2; and p63mapk (Erk3-related) at 18q12-21.

Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.

As the overall age of the population increases, so does the need for a better understanding of the aging process in each tissue. We have previously reported that aging is associated with loss of dopamine β-hydroxylase levels in the retina.1 Recently, we have shown that increasing age in rats also produced increased tyrosine phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), a dysfunctional electroretinogram (ERG), and increased apoptosis in retinal lysates.2 Both IGF-1R and vascular endothelial cell growth factor (VEGF) are increased in the aging rat retina, suggesting that normal aging may promote the vascular growth of normally quiescent tissue.3 Occurring concurrently with the increase in VEGF and IGF-1R is an increase in β-1-adrenergic receptor levels, likely because of denervation sensitivity in response to reduced norepinephrine.1 It is unclear whether the changes in IGF-1R and VEGF are related to this loss of norepinephrine in aging. The role of IGF-1R in the retina is still unclear. IGF-1 receptor (IGF-1R) mutations allow for increased life spans in multiple organisms,4,5 which may occur because these animals are resistant to cancer.6,7 IGF-1R may promote cancer and other age-related diseases through its ability to activate both the mitogen activated kinase pathway and the phosphatidylinositol-3-kinase cascade.8,9 In the retina, IGF-1R signaling has been shown to be involved with neovascularization in retinopathy of prematurity,10 diabetic retinopathy,11,12 normal development,13 and normal aging.2 However, the regulation of IGF-1R in the retina is unknown. Insulin-like growth factor binding proteins (IGFBPs) regulate circulating IGF-I bioavailability. All IGFBPs function at some level to regulate the delivery of IGF molecules to local tissues,14 with the predominating IGFBP species determined by cell type and local conditions. Both IGFBP-3 and IGFBP-5 bind the extracellular matrix,14 which provides a physiological “sink” in which to store these IGFBPs. The extracellular matrix is disrupted in normal aging15,16; hence, protein levels of IGFBP-3 and IGFBP-5 may be decreased because of abnormal extracellular matrix. Work in RPE samples of elderly humans has shown that aging can regulate the expression of IGFBP-2 in vitro.17 In other work, all six of the IGFBPs have been localized in the RPE in culture.18 Retinal Muller cells in culture express IGFBP-2, -3, -4, and -6.19 IGFBP-3 is expressed on the surface and in the cytoplasm of human retinal endothelial cells.20 We have previously shown that both retinal Muller cells and endothelial cells have β-adrenergic receptors and, therefore, may regulate IGFBP levels in the retina.21–23 In addition to the IGFBPs, limited work has demonstrated that β-adrenergic receptors may regulate IGF-1 levels and signaling. Evidence from β-2-adrenergic receptor knockout mice demonstrated that loss of β-2-adrenergic receptors in astrocytes increases IGF1R gene expression.24 Whether these findings can be extended to the retina is not known because most work on IGF-1R signaling has investigated the ability of IGF-1R to regulate β-adrenergic receptor signaling.25 Our goal was to determine whether β-adrenergic receptors can regulate IGF-1R levels in the rat retina. Given that aging is associated with a loss of norepinephrine, we hypothesized that the blockade of β-adrenergic receptors in young rats will induce changes in the retina similar to those noted in aging, specifically increased IGF-1R signaling. To test our hypothesis, we placed osmotic pumps containing saline, phentolamine (nonselective α-adrenergic receptor antagonist), or propranolol (nonselective β-adrenergic receptor antagonist) into 8-week-old rats for 21 days of systemic delivery of the drug. The doses and time course for treatment were based on previous work with this technique, which demonstrated that propranolol at 1 mg/d for 21 days could elicit histologic changes in the rat retina and choroid.26 We evaluated IGF-1R signaling and electroretinographic findings of rats in each treatment group to measure whether the blockade of adrenergic receptor signaling in youth could produce changes similar to those that occur during normal aging of the retina.

Purpose PAX6 heterozygosity (PAX6(+/-)) causes aniridia and aniridia-related keratopathy (ARK) in humans, but the pathway from gene dosage deficiency to clinical disease has not been fully characterized. Recently, the authors suggested a model of a chronic wound state exacerbated by oxidative stress, showed the barrier function of Pax6(+/-) corneas is compromised and suggested Pax6(+/-) corneas show the molecular signature of a perpetual wound-healing state. Methods Pax6(+/-) mice were used as a model for Pax6-related corneal diseases and in vivo wound-healing assays. Immunohistochemistry and electron microscopy analyses were performed on mutant and wounded corneas. Results This work reports defects in keratin, desmoplakin, and actin-based cytoskeletal structures in Pax6(+/-) cells. During wild-type corneal reepithelialization, cell fissures and desquamation, intracellular vesicles, intercellular gaps, and filopodialike structures were apparent, similar to the phenotypes seen in "unwounded" Pax6(+/-) corneal epithelia. Pax6(+/-) cells and wounded wild-type cells showed changed patterns of desmoplakin and actin localization. Protein oxidation and ERK1/2 and p38 MAPK phosphorylation were barely detected in the basal cells of intact wild-type corneal epithelia, but they were found in basal wild-type cells near the wound edge and throughout Pax6(+/-) corneal epithelia. Conclusions These data show that cell junctions and cytoskeleton organization are dynamically remodeled in vivo by wounding and in Pax6(+/-) corneas. This apparent wound-healing phenotype contributes to the clinical aspects of ARK.

PURPOSE To investigate the differential roles of matrix metalloproteinase (MMP)-9 and MMP-2 in the proliferation or differentiation of retinoblastoma cells. METHODS Cell proliferation assay with an MMP-9 inhibitor and cell viability assay with an MMP-2 inhibitor were performed in retinoblastoma cells with 5 ng/mL fibroblast growth factor 2 for proliferation, 0.1% bovine serum albumin for differentiation, or reverse transcriptase-polymerase chain reaction (RT-PCR) for MMP-9, MMP-2, and their tissue inhibitors TIMP-1 and TIMP-2. Immunohistochemistry for MMP-2 and nm23 was performed using an experimental model of retinoblastoma. With the use of an MMP-2 inhibitor, Western blot analysis was performed for neurofilament, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and phospho-ERK 1/2, and neurite length was measured in differentiated retinoblastoma cells. RESULTS With the proliferation of retinoblastoma cells, MMP-9 expression was upregulated without alteration of MMP-2, TIMP-1, or TIMP-2. However, proliferation was not affected by the inhibition of MMP-9 activity. Interestingly, only MMP-2 expression, colocalized with differentiated cells in retinoblastoma tissue, was significantly increased in the differentiation of retinoblastoma cells. Inhibition of MMP-2 activity did not affect cellular viability but attenuated neurite outgrowth and neurofilament expression of differentiated retinoblastoma cells, which was mediated through the suppression of ERK 1/2 activation. CONCLUSIONS The authors suggest that differential expression of MMP-9 and -2 could reflect biological features, such as proliferation and differentiation, of retinoblastoma cells. In particular, MMP-2 could be directly involved in the regulation of differentiation of retinoblastoma cells. Therefore, therapeutic targeting to MMP-2 may prove useful for reducing malignancy through the differentiation of retinoblastoma cells.

The Raf-1 proto-oncogene protein kinase can be phosphorylated and activated after stimulation of cells with insulin and a variety of other growth factors and mitogens. We recently presented evidence that insulin and certain other growth factors activated one or more Raf-1 kinase kinase activities (Lee, R.M., Rapp, U. R., and Blackshear, P.J. (1991) J. Biol. Chem. 266, 10351-10357). In the present study, four peaks of Raf-1 kinase kinase activity were identified after anion-exchange chromatography of cell lysates, and two of these were activated by insulin. Further chromatographic characterization of these two peaks of insulin-activated kinase activity indicated that they contained three apparently distinct kinase activities. Two of these activities comigrated with immunoreactive extracellular signal-regulated kinases (ERK) 1 and 2 (mitogen-activated protein kinase) through three different chromatographic separations. Both ERK1 and ERK2 phosphorylated Raf-1 with reasonably high affinity (Km for ERK1 = 90 nM; Km for ERK2 = 120 nM), and produced similar, complex phosphopeptide maps; both kinases also phosphorylated myelin basic protein. The third kinase activity also phosphorylated Raf-1 and myelin basic protein but did not comigrate exactly with either immunoreactive ERK1 or ERK2. We conclude that two and possibly three insulin-activated Raf-1 kinase kinases are members of the ERK family.

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.