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M H Cobb

Publications and source records attributed to M H Cobb.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dual MAP kinase pathways mediate opposing forms of long-term plasticity at CA3-CA1 synapses.

Although the function of the p42/p44 mitogen-activated protein (MAP) kinase pathway in long-term potentiation at hippocampal CA3-CA1 synapses has been well described, relatively little is known about the importance of the p38 MAP kinase pathway in synaptic plasticity. Here we show that the p38 MAP kinase pathway, a parallel signaling cascade activated by distinct upstream kinases, mediates the induction of metabotropic glutamate receptor-dependent long-term depression at CA3-CA1 synapses. Thus, two parallel MAP kinase pathways contribute to opposing forms of long-term plasticity at a central synapse.

Animals↗
PubMed1999

The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo.

An ERK2-binding site at the N terminus of MEK1 was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the phosphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 with ERK2 and Raf-1. Deletion of the binding site from MEK1 reduced its phosphorylation by ERK2, but had no effect on its phosphorylation by p21-activated protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the binding site inhibited MEK1 phosphorylation by ERK2. However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protein phosphorylation by ERK2. Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immunoprecipitate with ERK2, and to stimulate ERK2 activation in transfected cells, but it did not alter the association with endogenous Raf-1. Using ERK2-p38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was localized to its N terminus.

Amino Acid Sequence↗
PubMed1999

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.

Animals↗
PubMed1999

Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase.

p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.

Heterotrimeric GTP-Binding Proteins↗
PubMed1999

Isolation of the protein kinase TAO2 and identification of its mitogen-activated protein kinase/extracellular signal-regulated kinase kinase binding domain.

We previously reported the cloning of the thousand and one-amino acid protein kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase sterile 20 protein. Here we report the complete sequence and properties of a related rat protein kinase TAO2. Like TAO1, recombinant TAO2 selectively activated mitogen-activated protein/extracellular signal-regulated kinase kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells. To examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was localized to an approximately 135-residue sequence just C-terminal to the TAO2 catalytic domain. In vitro this MEK binding domain associated with MEKs 3 and 6 but not MEKs 1, 2, or 4. Using chimeric MEK proteins, we found that the MEK N terminus was sufficient for binding to TAO2. Catalytic activity of full-length TAO2 enhanced its binding to MEKs. However, neither the autophosphorylation of the MEK binding domain of TAO2 nor the activity of MEK itself was required for MEK binding. These results suggest that TAO proteins lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets.

Amino Acid Sequence↗
PubMed1999

Phosphorylation of MAP kinases by MAP/ERK involves multiple regions of MAP kinases.

Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N- and C-terminal domains.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Lipopolysaccharide-induced tumor necrosis factor-alpha promoter activity is inhibitor of nuclear factor-kappaB kinase-dependent.

The adverse effects of lipopolysaccharide (LPS) are primarily mediated by tumor necrosis factor-alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated both transcriptionally and post-transcriptionally. Transcriptional regulation of the TNF-alpha gene is dependent on nuclear factor-kappaB (NF-kappaB). We examined the signaling pathways involved in the regulation of NF-kappaB that lead to TNF-alpha promoter activity. We determined a role for one or both of the recently identified inhibitor of NF-kappaB kinases, IkappaB kinase-1 and IkappaB kinase-2, in LPS induction of an NF-kappaB reporter and of TNF-alpha promoter activity. IkappaB kinase activation is one of the earliest signaling events known to be induced by LPS. Furthermore, our results suggest roles for the IkappaB kinases NF-kappaB-inducing kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 in the regulation of IkappaB kinase-2, as well as in LPS-induced TNF-alpha transcription.

Animals↗
PubMed1999

MEKK1 interacts with alpha-actinin and localizes to stress fibers and focal adhesions.

Mitogen-activated protein (MAP) kinases orchestrate the effects of many extracellular stimuli on cells. The serine/threonine protein kinase MEKK1 is an upstream activator of the MAP kinases c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), extracellular signal-regulated kinase (ERK), and p38 as well as NF-kappa B. In a yeast two-hybrid interaction screen to identify proteins that bind to an N-terminal fragment of MEKK1 (amino acids 1-719), the actin-crosslinking protein alpha-actinin was identified as a MEKK1-binding protein. Over-expressed MEKK1 co-immunoprecipitated with alpha-actinin in cell lysates. Both endogenous and over-expressed MEKK1 colocalized with alpha-actinin along actin stress fibers and at focal adhesions. Residues 221-559 of MEKK1 bound to purified alpha-actinin in vitro, indicating that the interaction is direct, and this fragment localized to actin filaments in cells. MEKK1 kinase activity was not required for association with actin filaments, because a catalytically inactive mutant of MEKK1 (MEKK1 D1369A) localized to stress fibers. These results provide strong evidence for the interaction between MEKK1 and alpha-actinin. Thus, restriction of the kinase to the actin cytoskeleton may serve to regulate its specificity towards downstream targets.

3T3 Cells↗
PubMed1999

MAP kinase pathways.

MAP kinases help to mediate diverse processes ranging from transcription of protooncogenes to programmed cell death. More than a dozen mammalian MAP kinase family members have been discovered and include, among others, the well studied ERKs and several stress-sensitive enzymes. MAP kinases lie within protein kinase cascades. Each cascade consists of no fewer than three enzymes that are activated in series. Cascades convey information to effectors, coordinates incoming information from other signaling pathways, amplify signals, and allow for a variety of response patterns. Subcellular localization of enzymes in the cascades is an important aspect of their mechanisms of action and contributes to cell-type and ligand-specific responses. Recent findings on these properties of MAP kinase cascades are the major focus of this review.

Animals↗
PubMed1999

Scaffolding and protein interactions in MAP kinase modules.

MAP kinases are a family of protein kinases that are ubiquitously expressed and play roles in most signal transduction pathways. They are activated within protein kinase cascades consisting of at least three kinases acting in series. In many, if not all cases, the three-kinase cascade, conveniently referred to as a MAP kinase module, is organized on scaffolds with a variety of forms and functions. This review discusses similarities and differences in scaffolding proteins and mechanisms in yeast, flies, worms and mammals.

Animals↗
PubMed1998

Isolation of TAO1, a protein kinase that activates MEKs in stress-activated protein kinase cascades.

Several components of the budding yeast pheromone-response pathway are conserved in mammalian mitogen-activated protein (MAP) kinase pathways. Thus, we used degenerate oligonucleotides derived from the sequence of the Saccharomyces cerevisiae protein kinase Ste20p to amplify related sequences from the rat. One of these sequences was used to clone a rat Ste20p homolog, which we called TAO1 for its one thousand and one amino acids. Northern analysis shows TAO1 is highly expressed in brain, as is a homolog TAO2. Recombinant TAO1 was expressed and purified from Sf9 cells. In vitro, it activated MAP/extracellular signal-regulated protein kinase (ERK) kinases (MEKs) 3, 4, and 6 of the stress-responsive MAP kinase pathways, but not MEK1 or 2 of the classical MAP kinase pathway. TAO1 activated MEK3 but not MEK4 or MEK6 in transfected cells. MEK3 coimmunoprecipitated with TAO1 when they were expressed in 293 cells. In addition, immunoreactive MEK3 endogenous to Sf9 cells copurified with TAO1 produced from a recombinant baculovirus. The activation of and binding to MEK3 by TAO1 implicates TAO1 in the regulation of the p38-containing stress-responsive MAP kinase pathway.

Amino Acid Sequence↗
PubMed1998

Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation.

PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.

Cell Adhesion↗
PubMed1998

A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation.

BACKGROUND: Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS: To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS: Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.

3T3 Cells↗
PubMed1998

Structural basis of inhibitor selectivity in MAP kinases.

BACKGROUND: The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS: We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS: The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Adenosine Triphosphate↗
PubMed1998

The MEK1 proline-rich insert is required for efficient activation of the mitogen-activated protein kinases ERK1 and ERK2 in mammalian cells.

MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

Animals↗
PubMed1998

Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation.

The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

Animals↗