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Anaplastic large-cell lymphoma (ALCL) cells overexpress CD30 on their cell surface, show increased levels of activated Erk1/2 and of JunB; participating JunB in the proliferative capacity of these lymphomas. Here, we show that ALCL lymphoma cells also present high expression levels of the proto-oncogenic Cot (MAP3K8). Using pharmacological drugs as well as the RNA interference technique we show that Cot protein is responsible for the constitutive Erk1/2 activation in the ALCL lymphoma cells, SUDHL-1. Besides, inhibition of Cot activity reduces the number of cell divisions which is achieved, at least in part, by the control that Cot exercises on the activation state of p70 S6K and on the expression levels of JunB. Since Cot represents an alternative mode, independently of RAF, to activate Erk1/2, all these data strongly suggest that molecular targeting of Cot may be a potential new specific strategy for ALCL lymphomas therapy, without the fully disturbance of the Erk1/2 function. © 2011., This work was supported by a grant from the Ministerio de Ciencia e Innovación (SAF 2008-00819) and Mutua Madrileña.

The MAPKKK8 Cot/tpl-2, identified as an oncogene (Cot-T), participates in the intracellular signaling activated by members of the TLR and TNFalpha receptor superfamilies. Here we demonstrate that Cot promotes cell migration by regulating different steps involved in this process, such as cell adhesion and metalloproteinase activity. Indeed, Cot also regulates the cytoskeleton and Cot-T overexpression provokes the polarization of microtubules and the loss of stress fibers. Moreover, and in accordance with the increased Rac-GTP levels observed, Cot-T overexpressing cells develop more lamellipodia than control cells. Conversely, depletion of endogenous Cot increases the formation of stress fibers which is correlated with the high levels of Rho-GTP observed in these cells. In addition, the increase in COX2 expression and the activation of Erk1/2 regulated by Cot are essential for the induction of cell migration. Together, these data provide evidence of a new role for both proto-oncogenic and oncogenic Cot.

Background X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. Objective The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. Methods Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-α and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. Results We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-α and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. Conclusion Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. Clinical implications These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models.

COT/Tpl-2 proto-oncogene encodes a serine/threonine kinase implicated in cellular activation. In this study we have identified the human COT gene promoter region and three different human COT transcripts. These transcripts, with the same initiation site, display heterogeneity in their 5′ untranslated regions and in their subcellular localization. Activation of Jurkat T cells with either calcium ionophore A23187 or αCD3 and a phorbol ester increases the levels of the different COT transcripts. Analysis of the 5′ flanking region of the human COT gene reveals a unique transcription initiation site and a TATA element 20 nucleotides upstream. Transient expression of COT promoter constructs containing a reporter gene indicates that the transcriptional activity of the 5′ flanking region of the COT gene is regulated by T cell-activating signals. Cotransfection of a dominant negative version of SEK-2 abolishes the inducible transcriptional activity ofCOT promoter, indicating that the inducible expression of the COT gene by T cell activating signals is mediated by the JNK/SAPK signal transduction pathway. All these data indicate stringent regulation of COT kinase proto-oncogene expression.

Cot kinase is a protein serine/threonine kinase, classified as a mitogen-activated protein kinase kinase kinase, implicated in T lymphocyte activation. Here we show that an increase in Cot kinase expression promotes tumor necrosis factor-alpha (TNF-alpha) production in Jurkat T cells stimulated by soluble anti-CD3 or by low concentrations of phorbol 12,13-dibutyrate (PDBu) and calcium ionophore. Overexpression of Cot kinase in Jurkat cells activates TNF-alpha gene expression. Cot kinase promotes TNF-alpha promoter activation to a similar extent as calcium ionophore and PDBu or soluble anti-CD28 and PDBu. Neither phorbol esters nor calcium ionophore can replace Cot kinase on TNF-alpha promoter-driven transcription. Expression of a dominant negative form of Cot kinase inhibits TNF-alpha promoter activation induced by stimulation with either calcium ionophore and PDBu, soluble anti-CD28 and PDBu, or soluble anti-CD3 and PDBu. TNF-alpha promoter-driven transcription by Cot kinase is partially mediated by MAPK/ERK kinase and is cyclosporin A-resistant. Cot kinase increases at least the AP-1 and AP-2 response elements. These data indicate that Cot kinase plays a critical role in TNF-alpha production.

A phospho-oligosaccharide, whose production is stimulated by insulin, modulated the activity of partially purified casein kinase II. Whereas at 2 microM the phospho-oligosaccharide stimulated casein kinase II 1.3-fold, higher concentrations of this molecule were inhibitory. 50% inhibition of the enzyme was obtained at 15 microM phospho-oligosaccharide. This biphasic effect of the phospho-oligosaccharide on casein kinase II activity was observed using as substrate both casein or the specific peptide for casein kinase II, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. The effect of the phospho-oligosaccharide on casein kinase II was still observed after gel filtration. Deamination of the phospho-oligosaccharide with nitrous acid abolished both the activation and the inhibition of casein kinase II. The glycophospholipid precursor of the phospho-oligosaccharide did not affect casein kinase II activity. Moreover, modulation of casein kinase II activity was not observed with other compounds structurally related to the phospho-oligosaccharide, when used in the micro-molar range. In conclusion, the present results indicate that the phospho-oligosaccharide that mimics and might mediate some of the actions of insulin modulates casein kinase II activity in vitro.

The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes (see ref. 4). We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phosphooligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations.

Corrigendum Corrigendum to ‘‘Involvement of Cot activity in the proliferation of ALCL lymphoma cells’’ [Biochem. Biophys. Res. Commun. 411 (2011) 655–660] Margarita Fernandez ⇑, Rebeca Manso, Flavia Bernaldo de Quiros, Pilar Lopez, Antonio Martin-Duce, Susana Alemany Instituto de Investigaciones Biomedicas ‘‘Alberto Sols’’, CSIC-UAM, Madrid and Departamento de Bioquimica, Facultad de Medicina de la Universidad Autonoma de Madrid, Spain

In the presence of ( 3 H-methyl)-S-adenosyl-L-methionine Di ctyostelium discoideum cell homogenates incorporate ( 3 H-methyl) groups into mono- and dimethyl-phosphatidylethanolamine and phosphatidylcholine. The addition of bovine brain calmodulin enhances lipid transmethylation and an antiserum against rat brain calmodulin inhibits the reaction. EGTA and chlorpromazine, an inhibitor of calmodulin action, inhibit phospholipid methylation. Based on these results we propose that this reaction is modulated by calmodulin.

Rat liver S-adenosyl-L-methionine synthetase exists in two forms which are, respectively, a dimer and a tetramer of an Mr 48,500 subunit. The high-molecular-mass form is converted into the low-molecular-mass form by incubation with 1.4 M LiBr. The kinetic properties of the low-molecular-mass form obtained by LiBr treatment are the same as those obtained with the low-molecular-mass S-adenosyl-L-methionine synthetase form purified from rat liver cytosol. These results demonstrate that the differences in specific activity and regulatory properties of the high-molecular-mass and the low-molecular-mass S-adenosyl-L-methionine synthetase forms are due to their different polymeric states.