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A R Nebreda

Publications and source records attributed to A R Nebreda.

At least 37 records · Page 2Linked to original sources
PubMed1998

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.

Animals↗
PubMed1997

Reactivating kinase/p38 phosphorylates tau protein in vitro.

Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer-diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated tau protein. Tau from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate tau in vitro; those that require or accept prolines include GSK3 and members of the mitogen-activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase-beta/JNK. In this report, we show that another member of the MAP kinase family, the stress-activated kinase p38/RK, can phosphorylate tau in vitro. Western blots with phosphorylation-sensitive antibodies showed that p38, like ERK2 and SAP kinase-beta/JNK, phosphorylated tau at sites found phosphorylated physiologically (Thr181, Ser202, Thr205, and Ser396) and also at Ser422, which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to tau hyperphosphorylation during the formation of PHFs, and hence, to the development of tau pathology.

Alzheimer Disease↗
PubMed1996

Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6.

Amino Acid Sequence↗
PubMed1996

Cellular stresses and cytokines activate multiple mitogen-activated-protein kinase kinase homologues in PC12 and KB cells.

The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.

Amino Acid Sequence↗
PubMed1995

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.

Animals↗
PubMed1995

Activation of the MAP kinase homologue RK requires the phosphorylation of Thr-180 and Tyr-182 and both residues are phosphorylated in chemically stressed KB cells.

A MAP kinase homologue, termed the reactivating kinase (RK), lies in a signalling pathway which mediates cellular responses to stress. Here we demonstrate that the stress-induced activation of the RK in human KB cells is accompanied by the phosphorylation of Thr-180 and Tyr-182, and that the phosphorylation of both residues is required for the activation of this enzyme.

Amino Acid Sequence↗
PubMed1994

A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.

MAPK-activated protein kinase-2 (MAPKAP kinase-2) is activated in vitro by the p42 and p44 isoforms of MAPK (p42/p44MAPK). In several cell lines, however, MAPKAP kinase-2 is activated by sodium arsenite, heat shock, or osmotic stress and not by agonists that activate p42/p44MAPK. We have identified a MAPK-like enzyme that acts as a MAPKAP kinase-2 reactivating kinase (RK). RK is recognized by an antiserum raised against a Xenopus MAPK (Mpk2), which is most similar to HOG1 from S. cerevisiae. We also identified a RK kinase (RKK) on the basis of its ability to activate either RK or a GST-Mpk2 fusion protein. The RKK, RK, and MAPKAP kinase-2 constitute a new stress-activated signal transduction pathway in vertebrates that is distinct from the classical MAPK cascade.

Amino Acid Sequence↗
PubMed1993

The protein kinase mos activates MAP kinase kinase in vitro and stimulates the MAP kinase pathway in mammalian somatic cells in vivo.

The mos protooncogene encodes a serine/threonine protein kinase that is only expressed at significant levels in germ cells. Recombinant malE-mos protein (Xenopus mos protooncogene fused in frame to the maltose binding protein of E. coli) activates MAP kinase in cell-free extracts prepared from Xenopus oocytes and eggs. Here we show that malE-mos immunoprecipitates from Xenopus extracts phosphorylate and activate MAP kinase kinase in vitro, indicating that mos can function as a MAP kinase kinase kinase. Moreover, ectopic expression of mos in mammalian somatic cells, that lack any endogenous mos protein, triggers the activation of MAP kinase in vivo. These results identify the mos protooncogene as a direct activator of the MAP kinase pathway, with the potential to activate this kinase cascade even in cells where normally there is no expression of mos.

3T3 Cells↗
PubMed1993

Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes.

Mitogens and growth factors acutely stimulate glucose transport in all cells to supply energy for their growth and division, but little is known about the signalling mechanism by which these agonists promote sugar uptake. Here we show that the transport of deoxyglucose and 3-O-methylglucose into Xenopus laevis oocytes is stimulated about 2.5-fold when mitogen-activated protein kinase (MAP kinase) is microinjected into these oocytes. We also demonstrate that microinjection of the proto-oncogene product c-Mos (an activator of MAP kinase kinase, which activates MAP kinase in Xenopus oocytes), and purified MAP kinase kinase produce similar increases in deoxyglucose transport. Since the activation of MAP kinase is a general response to almost all mitogens and growth factors, we propose that one of its downstream effects is the stimulation of glucose-transport activity.

3-O-Methylglucose↗
PubMed1993

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.

ATP-Binding Cassette Transporters↗
PubMed1993

p21ras-induced meiotic maturation of Xenopus oocytes in the absence of protein synthesis: MPF activation is preceded by activation of MAP and S6 kinases.

Microinjection of transforming p21ras into Xenopus oocytes caused a time-dependent increase in the level of total cell protein phosphorylation that culminated with germinal vesicle breakdown (GVBD). The same pattern of phosphorylation was observed in oocytes matured by either progesterone or insulin. Treatment with cycloheximide (CHX) completely blocked both GVBD and the associated de novo phosphorylations induced by the hormones, but did not abolish p21ras-induced maturation nor the occurrence of associated maturation promoting factor (MPF)-dependent and -independent phosphorylations. Thus, induction of GVBD by p21ras in the absence of protein synthesis correlated with the activation of cytosolic MPF-associated kinase activity similar in specificity on exogenous (histone H1) and endogenous (47 kDa and a 42 kDa proteins) substrates to the MPF activity of hormonally-matured oocytes. The injection of p21ras in the presence of CHX caused also activation of other kinase(s) proceeding MPF activation which were responsible for the phosphorylation of endogenous substrates including a 41 kDa protein and a 92 kDa protein kinase that comigrated, respectively, with bands recognized specifically by antibodies to MAP2 kinase and S6 kinase. The phosphorylation of those bands correlated also with the activation of cytosolic kinases acting specifically on myelin basic protein (MBP) and a S6-derived peptide as substrates. These results indicate that, in the absence of protein synthesis, p21ras is able to activate phosphorylation events leading to GVBD and suggest that this oncoprotein can participate in at least two separate pathways of MPF activation. We propose that the activation of MAP/MBP kinases and S6 kinases is an early effect of p21ras oncoproteins.

Animals↗
PubMed1992

Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active Ras.GTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP), insulin-treated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin.

3T3 Cells↗
PubMed1992

Induction of Xenopus oocyte meiotic maturation by the dbl oncogene product.

The dbl oncogene was originally identified by transfection of NIH3T3 cells with DNA from a human diffuse B-cell lymphoma. The dbl oncogene product is a cytoplasmic phosphoprotein distributed between the cytosolic and cytoskeletal matrix-associated membrane fractions. Nucleotide sequence analysis has indicated that the predicted dbl product is very hydrophilic with no detectable similarity to known oncogene products. We have more recently discovered that a region of dbl essential for its transforming activity shows significant sequence similarity to a yeast cell cycle gene, CDC24, which is involved in cell polarity and bud formation in the division cycle of Saccharomyces cerevisiae. This sequence similarity suggests a possible role for dbl in cell division. We report here that the dbl oncogene product is able to induce maturation of Xenopus oocytes. Germinal vesicle breakdown (GVBD) was observed when oocytes were microinjected with the soluble fraction of SF9 insect cells infected with a dbl recombinant baculovirus as well as with the in vitro-transcribed dbl mRNA. Moreover, extracts of oocytes microinjected with dbl mRNA showed activation of H1 histone kinase activity. These findings define a new biologic activity of the dbl product and provide the opportunity to analyse dbl interactions with other components of signaling pathways involved in oocyte maturation.

Animals↗
PubMed1991

Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes.

Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I (IGF-1). Expression of transfected ras oncogenes led to differentiation of these cells into adipocytes in the absence of externally added insulin or IGF-I. Cells transfected with normal ras genes or the tyrosine kinase trk oncogene did not differentiate. Transfection with a dominant inhibitory ras mutant resulted in inhibition of differentiation. Exposure of untransfected 3T3-L1 cells to insulin stimulated formation of the active Ras.GTP complex. These observations indicate that Ras proteins participate in signal transduction pathways initiated by insulin and IGF-I in these cells.

Adipose Tissue↗
PubMed1991

The ras oncoprotein and M-phase activity.

The endogenous mos proto-oncogene product (Mos) is required for meiotic maturation. In Xenopus oocytes, the ras oncogene product (Ras) can induce meiotic maturation and high levels of M-phase--promoting factor (MPF) independent of endogenous Mos, indicating that a parallel pathway to metaphase exists. In addition, Ras, like Mos and cytostatic factor, can arrest Xenopus embryonic cell cleavage in mitosis and maintain high levels of MPF. Thus, in the Xenopus oocyte and embryo systems Ras functions in the M phase of the cell cycle. The embryonic cleavage arrest assay is a rapid and sensitive test for Ras function.

Animals↗
PubMed1991

Induction by NGF of meiotic maturation of Xenopus oocytes expressing the trk proto-oncogene product.

The effect of nerve growth factor (NGF) was assessed in Xenopus oocytes expressing the human trk proto-oncogene product, p140prototrk. Oocytes injected with trk messenger RNA expressed polypeptides recognized by antibodies to the trk gene product. Exposure of these oocytes to nanomolar amounts of NGF resulted in specific surface binding of 125I-labeled NGF, tyrosine phosphorylation of p140prototrk, and meiotic maturation, as determined by germinal vesicle breakdown and maturation promoting factor (p34cdc2) kinase activation. Thus the trk proto-oncogene product can act as a receptor for NGF in a functionally productive manner.

Animals↗
PubMed1991

Nucleotide sequence of the exo-1,3-beta-glucanase-encoding gene, EXG1, of the yeast Saccharomyces cerevisiae.

The nucleotide (nt) sequence of the Saccharomyces cerevisiae gene (EXG1) encoding extracellular exo-1,3-beta-glucanases (EXG) I and II was determined. An open reading frame of 1344 bp codes for a 448-amino acid (aa) polypeptide, with a calculated Mr of 51,307, which contains two potential N-glycosylation sites. The EXG1 DNA hybridizes to a 1.7-kb transcript whose 5' end maps to a position 98 bp upstream from the site of initiation of protein synthesis. Comparison of the N-terminal aa sequence deduced from the nt sequence with that of the purified EXGII revealed the existence of an extra 40-aa peptide in the precursor protein containing a Lys-Arg peptidase-processing site at the junction with the mature, extracellular form. The N-terminal region of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. The Mr of the mature EXG polypeptide deduced from the nt sequence is 46,385. The 5'- and 3'-flanking regions of the EXG1 gene have structural features in common with other yeast genes.

Amino Acid Sequence↗