Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

Sustained mitogen-activated protein kinase activation is induced by transforming erbB receptor complexes.

We used a genetic approach to characterize features of mitogen-activated protein kinase (MAPK) activation occurring as a consequence of expression of distinct erbB receptor combinations in transformed human cells. Kinase-deficient erbB proteins reduced epidermal growth factor (EGF)-induced tyrosine phosphorylation of endogenous Shc proteins and also reduced immediate and sustained EGF-induced ERK MAPK activities in human glioblastoma cells, although basal ERK MAPK activities were unaffected. Basal and EGF-induced JNK and p38 MAPK kinase activities were equivalent in parental cancer cells and EGFR-inhibited subclones. When ectopically overexpressed in murine fibroblasts and human glioblastoma cells, a constitutively activated human EGF receptor oncoprotein (deltaEGFR) induced EGF-independent elevation of basal ERK MAPK activity. Basal JNK MAPK kinase activity was also specifically induced by deltaEGFR, which correlated with increased phosphorylation of a 54-kDa JNK2 protein observed in deltaEGFR-containing cells. The JNK activities in response to DNA damage were comparably increased in cells containing wildtype EGFR or deltaEGFR. Consistent with the notion that transforming erbB complexes induce sustained and unregulated MAPK activities, coexpression of p185(neu) and EGFR proteins to levels sufficient to transform murine fibroblasts also resulted in prolonged EGF-induced ERK in vitro kinase activation. Transforming erbB complexes, including EGFR homodimers, deltaEGFR homodimers, and p185(neu)/EGFR heterodimers, appear to induce sustained, unattenuated activation of MAPK activities that may contribute to increased transformation and resistance to apoptosis in primary human glioblastoma cells.     

Role of interleukin (IL)-2 receptor beta-chain subdomains and Shc in p38 mitogen-activated protein (MAP) kinase and p54 MAP kinase (stress-activated protein Kinase/c-Jun N-terminal kinase) activation. IL-2-driven proliferation is independent of p38 and p54 MAP kinase activation

We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rbeta chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rbeta chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2.     

Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90^rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90^rsk. The MAP kinase p42^mpk1 can associate with p90^rsk in G₂-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90^rsk mutant named D2 constitutively interacts with p42^mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42^mpk1 docking site. D2 expression uncouples the activation of p42^mpk1 and p34^cdc2/cyclin B in response to progesterone but does not prevent signaling through p90^rsk. Instead, D2 interferes with a p42^mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42^mpk1 and Plx1 during oocyte maturation is independent of p34^cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42^mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.     

p66Shc-dependent apoptosis requires Lck and CamKII activity

p66Shc, an adaptor molecule which enhances reactive oxygen species (ROS) production by mitochondria, promotes T-cell apoptosis by inducing mitochondrial dysfunction and impairing Ca²⁺ homeostasis. We have addressed the potential role of Lck, a kinase which has been implicated in T-cell apoptosis induced by a number of stimuli, in the proapoptotic activity of p66Shc. Lck deficiency in Jurkat T cells overexpressing p66Shc leads to impaired apoptotic responses to supraphysiological increases in [Ca²⁺]_c. This defect could be rescued by reconstitution of Lck expression, indicating that Lck is required for p66Shc-dependent apoptosis. Furthermore, p66Shc phosphorylation on serine 36 (S36), an event on which the proapoptotic function of p66Shc depends, requires Lck. p66Shc-dependent mitochondrial dysfunction, altered Ca²⁺ homeostasis and S36 phosphorylation require moreover the activity of CaMKII, a Ca²⁺/calmodulin-dependent kinase known to be implicated in the proapoptotic activity of Lck in T cells. The results suggest that increases in [Ca²⁺]_c lead to CaMKII activation and subsequent Lck-dependent p66Shc phosphorylation on S36. This event causes both mitochondrial dysfunction and impaired Ca²⁺ homeostasis, which synergize in promoting Jurkat T-cell apoptosis.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

Coupling of Serotonin 5-HT1B Receptors to Activation of Mitogen-Activated Protein Kinase (ERK-2) and p70 S6 Kinase Signaling Systems

Abstract: Little is known about the coupling of serotonin 5-HT_1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5-HT_1B receptors of p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) ERK-2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5-HT_1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5-HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC₅₀ of 20 n M . Reflecting increased density of receptors, 5-HT caused a greater than eightfold activation of ERK-2 in transfected cells with an EC₅₀ of 2 n M . 5-HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC₅₀ for 5-HT was fourfold lower in transfected cells. The coupling of 5-HT_1B receptors to ERK-2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3-kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK-2, was also inhibited by rapamycin. These findings demonstrate that 5-HT_1B receptors couple to ERK-2 and p70 S6 kinase through overlapping, but nonidentical, pathways.     

Analyses of the effects of Rck2p mutants on Pbs2p<Superscript>DD</Superscript>-induced toxicity in<Emphasis Type="Italic"> Saccharomyces cervisiae</Emphasis> identify a MAP kinase docking motif,and unexpected functional inactivation due to acidic substitution of T379

Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2p^DD from a GAL1 -driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2p^DD. Rck2p kinase activity is required for the effect, because Rck2p(487–610), as well as full-length Rck2p, is toxic with Pbs2p^DD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR⁵⁸⁹R⁵⁹⁰KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2p^DD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2 ^DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2p^DD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2 strain to allow some cells to survive PBS2 ^DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.Communicated by M. Collart     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.     

Hydrogen Peroxide Enhances Signal-Responsive Arachidonic Acid Release from Neurons: Role of Mitogen-Activated Protein Kinase

Abstract: Hydrogen peroxide (H₂O₂) is a potent stimulator of signal-responsive phospholipase A₂ (PLA₂) in vascular smooth muscle and cultured endothelial cells. We investigated whether H₂O₂ plays a similar regulatory role in neurons. H₂O₂ did not stimulate a release of arachidonic acid from cultured neurons when applied alone but strongly enhanced the liberation of arachidonic acid evoked by maximally effective concentrations of either glutamate, the glutamate receptor agonist N-methyl-d -aspartate (NMDA), the muscarinic receptor agonist carbachol, the Na⁺-channel opener veratridine, or the Ca²⁺-ionophore ionomycin. The potentiating effects of H₂O₂ were strongly inhibited in the presence of the PLA₂ inhibitor mepacrine, suggesting that the site of action was within the signal responsive arachidonic acid cascade. The enhancing effect of H₂O₂ was not reversed by protein kinase C inhibitors (chelerythrine chloride or GF 109203X) nor was it mimicked by phorbol ester treatment. H₂O₂ alone strongly enhanced the levels of immunodetectable activated mitogen-activated protein kinase (activated MAP kinases ERK1 and ERK2) in a Ca²⁺-dependent manner and this effect was additive with increases in the levels of activated MAP kinase evoked by glutamate. The enhanced release of arachidonic acid, however, was not clearly reversed by the MAP kinase kinase (MEK) inhibitor PD 98059, although this treatment effectively abolished H₂O₂ activation of MAP kinase. Thus, MAP kinase activation and Ca²⁺-dependent arachidonic acid release are regulated by oxidative stress in cultured striatal neurons.     

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.     

Nociceptin (ORL-1) and μ-Opioid Receptors Mediate Mitogen-Activated Protein Kinase Activation in CHO Cells Through a Gi-Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist-Mediated Desensitization

Abstract: The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to G_i/G_o proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of β-adrenergic receptor kinase (βARKct), which specifically blocks Gβγ-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21^ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing μ-opioid receptor, [d -Ala²,N-Me-Phe⁴,Gly-ol]-enkephalin (DAMGO; a μ-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and μ-opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ-subunit of G_i, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and μ-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate μ-opioid receptor signaling in a cellular system.     

MEKK2 Kinase Association with 14-3-3 Protein Regulates Activation of c-Jun N-terminal Kinase

MEKK2 (MAP/ERK kinase kinase-2) is a serine/threonine kinase that belongs to the MEKK/STE11 family of MAP kinase kinase kinases (MAP(3)Ks). MEKK2 integrates stress and mitogenic signals to the activation of NF-κB, JNK1/2, p38, and ERK5 pathways. We have found that MEKK2 is regulated through a phosphorylation-dependent association with 14-3-3, a group of adapters that modulate dimerization and association between proteins. We found that MEKK2 was phosphorylated at Thr-283, which resulted in decreased activation loop phosphorylation at Ser-519 and consequently reduced activity. Mechanistically, we found that MEKK2 associated with inactive MEKK2 in the absence of 14-3-3 binding, which led to trans-autophosphorylation of Ser-519. Enforced binding with 14-3-3 reduced Ser-519 trans-autophosphorylation. Expression of T283A MEKK2 within a MEKK2^−/− background enhanced stress-activated c-Jun N-terminal kinase activity while elevating IL-6 expression, but also reduced ERK activation with a corresponding reduced proliferation rate. These results indicate that Thr-283 phosphorylation is an important regulatory mechanism for MEKK2 activation.     

P66Shc mediated ferritin degradation--a novel mechanism of ROS formation

Diallyl trisulfide (DATS) has been shown to induce the formation of reactive oxygen species (ROS) in prostate cancer cells, which was accompanied by a decrease in the ferritin protein level and an increase in the labile iron pool (LIP). However, the mechanism of the ferritin degradation has not been fully elucidated. In this paper we demonstrate that DATS-induced ROS formation depends on p66Shc. In cells stably expressing a dominant negative mutant of p66Shc (p66ShcS36A), DATS did not induce ROS formation. In addition, in cells expressing p66ShcS36A neither an increase in ferritin H degradation nor an increase in LIP were observed. Cells stably expressing p66ShcS36A also possess higher levels of ferritin H compared to PC-3 cells transfected with an empty vector. Moreover, DATS-induced G2/M arrest is completely abrogated in cells expressing p66ShcS36A. Mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or p66Shc knockout mouse have been used to evaluate if p66Shc involvement in DATS-induced signaling is cell specific. DATS induced G2/M arrest in WT MEFs but had no effect in the p66Shc^−/− cell line. Moreover, increases in LIP and ROS formation were significantly attenuated in p66Shc^−/− MEFs treated with DATS.     

Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan

The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A₂ (cPLA₂). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA₂ in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA₂ activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA₂ activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA₂ activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA₂ and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages.     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.