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M H Cobb

Publications and source records attributed to M H Cobb.

At least 19 recordsLinked to original sources
PubMed2001

kin-18, a C. elegans protein kinase involved in feeding.

TAO1 and TAO2 are recently described protein kinases whose initial characterization has placed them at the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase (MEKK) level of stress-responsive MAPK pathways. Because their physiological roles have not been identified, we sought to study their C. elegans homolog to learn more about their functions. kin-18 encodes a previously uncharacterized protein in C. elegans whose catalytic domain shares over 60% identity with TAO1 and TAO2. We demonstrate that KIN-18 is a protein of 120 kDa whose promoter is active in the pharynx and intestine of C. elegans. To learn more about TAO/KIN-18 function, we studied how expression of constitutively active forms of TAO1 or KIN-18 would affect the physiology of intact worms. Strains of C. elegans expressing active forms of TAO1 or KIN-18 exhibit altered pharyngeal electrophysiology as measured by electropharyngeogram. These worms grow more slowly and lay fewer eggs, phenotypes that could result from reduced feeding. We have also identified a C. elegans gene that encodes a protein kinase similar to mammalian MAPK/ERK Kinase (MEK) 4 whose promoter is active in the pharynx. It is phosphorylated by TAO1 in vitro and physically interacts with TAO1.

Amino Acid Sequence↗
PubMed2001

Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking.

Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.

Amino Acid Sequence↗
PubMed2001

Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2.

Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.

Amino Acid Sequence↗
PubMed2001

ERKs weigh in on ribosome mass.

In this issue of Molecular Cell, Stefanovsky et al. demonstrate that the activation of the ERK1/2 MAP kinases by growth factors leads to induction of ribosomal gene transcription, through a mechanism dependent on phosphorylation of the upstream binding factor (UBF). This provides a connection between growth factor signaling and increased translation.

DNA-Binding Proteins↗
PubMed2001

MAP kinases.

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Mitogen-Activated Protein Kinases↗
PubMed2001

Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions.

Mitogen-activated protein (MAP) kinases comprise a family of ubiquitous proline-directed, protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events including acute responses to hormones and major developmental changes in organisms. MAP kinases lie in protein kinase cascades. This review discusses the regulation and functions of mammalian MAP kinases. Nonenzymatic mechanisms that impact MAP kinase functions and findings from gene disruption studies are highlighted. Particular emphasis is on ERK1/2.

Animals↗
PubMed2000

MEKK1 binds raf-1 and the ERK2 cascade components.

Mitogen-activated protein (MAP) kinase cascades are involved in transmitting signals that are generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK), and a MEK kinase (MEKK). Protein-protein interactions within these cascades provide a mechanism to control the localization and function of the proteins. MEKK1 is implicated in activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ERK1/2 MAP kinase pathways. We showed previously that MEKK1 binds directly to JNK/SAPK. In this study we demonstrate that endogenous MEKK1 binds to endogenous ERK2, MEK1, and another MEKK level kinase, Raf-1, suggesting that it can assemble all three proteins of the ERK2 MAP kinase module.

Cell Line↗
PubMed2000

ERK5 and ERK2 cooperate to regulate NF-kappaB and cell transformation.

We have previously demonstrated an involvement of MEK5 and ERK5 in RafBXB-stimulated focus formation in NIH3T3 cells. We find here that MEK5 and ERK5 cooperate with the RafBXB effectors MEK1/2 and ERK1/2 to induce foci. To further understand MEK5-ERK5-dependent signaling, we examined potential MEK5-ERK5 effectors that might influence focus-forming activity. Consistent with results from our focus-formation assays, constitutively active variants of MEK5 and MEK1 synergize to activate NF-kappaB, and MEK5 and ERK5 are required for activation of NF-kappaB by RafBXB. The MEK5-ERK5 pathway is also sufficient to activate both NF-kappaB and p90 ribosomal S6 kinase. Our results support the hypothesis that NF-kappaB and p90 ribosomal S6 kinase are involved in MEK5-ERK5-dependent focus formation and may serve as integration points for ERK5 and ERK1/2 signaling.

3T3 Cells↗
PubMed2000

Uncoupling Raf1 from MEK1/2 impairs only a subset of cellular responses to Raf activation.

The Raf family of serine/threonine protein kinases is intimately involved in the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf effector proteins. However, accumulating evidence suggests that there are additional Raf substrates and that subsets of Raf-induced regulatory events are mediated independently of Raf activation of MEK1/2. To examine the possibility that there is bifurcation at the level of Raf in activation of MEK1/2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association uncouples Raf from activation of ERK1/2, induction of serum-response element-dependent gene expression, and induction of growth and morphological transformation. However, activation of NF-kappaB-dependent gene expression and induction of neurite differentiation were unimpaired. In addition, Raf-dependent activation of p90 ribosomal S6 kinase was only slightly impaired. These results support the hypothesis that Raf kinases utilize multiple downstream effectors to regulate distinct cellular activities.

3T3 Cells↗
PubMed2000

Stress pathway activation induces phosphorylation of retinoid X receptor.

Cellular stresses inhibit retinoid signaling, but the molecular basis for this phenomenon has not been revealed. Here, we present evidence that retinoid X receptor (RXR) is a substrate for both mitogen-activated protein kinase kinase-4 (MKK4/SEK1) and its downstream mediator c-Jun N-terminal kinase (JNK). MKK4/SEK1 and JNK recognized distinct features on RXR in the DE and AB regions, respectively. Phosphorylation by MKK4/SEK1 had profound effects on the biochemical properties of RXR, inhibiting the expression of genes activated by RXR-retinoic acid receptor complexes. Tyr-249 in the RXR DE region was required for the inhibitory effect of MKK4/SEK1. These effects were significantly reduced in MKK4/SEK1-null cells, indicating that MKK4/SEK1 is required for the suppression of retinoid signaling by stress. Findings presented here demonstrate that MKK4/SEK1 can directly modulate transcription by phosphorylating RXR, a novel MKK4/SEK1 substrate.

Animals↗
PubMed2000

Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1.

The p21-activated kinase (PAK1) is a serine-threonine protein kinase that is activated by binding to the Rho family small G proteins Rac and Cdc42hs. Both Rac and Cdc42hs have been shown to regulate the activity of the transcription factor NFkappaB. Here we show that expression of active Ras, Raf-1, or Rac1 in fibroblasts stimulates NFkappaB in a PAK1-dependent manner and that expression of active PAK1 can stimulate NFkappaB on its own. Similarly, in macrophages activation of NFkappaB as well as transcription from the tumor necrosis factor alpha promoter depends on PAK1. In these cells lipopolysaccharide is a potent activator of PAK1 kinase activity. We also demonstrate that expression of active PAK1 stimulates the nuclear translocation of the p65 subunit of NFkappaB but does not activate the inhibitor of kappaB kinases alpha or beta. These data demonstrate that PAK1 is a crucial signaling molecule involved in NFkappaB activation by multiple stimuli.

3T3 Cells↗
PubMed2000

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II.

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.

Amino Acid Sequence↗
PubMed2000

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2.

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (</=0.5 microM) is significantly lower than that of the measured K(m(MBP)) (4.2 +/- 0.8 microM). Furthermore, MBP binds to the ERK2 x ATP complex at least 1500-fold more tightly than does ERKtide (K(d(ERKtide)) >/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (</=1.2 s(-1)) versus that of the synthetic peptide (>/=56 s(-1)), from the ERK2 active site.

Adenosine Triphosphate↗
PubMed2000

Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338.

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.

Animals↗
PubMed2000

IL-1 receptor-associated kinase modulates host responsiveness to endotoxin.

Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.

Animals↗
PubMed2000

Dimerization in MAP-kinase signaling.

The stimulus-dependent nuclear localization of the extracellular-signal- regulated kinases ERK1 and ERK2 is required for many of their actions, including induction of neurites in PC12 cells and transformation of fibroblasts. Phosphorylation of ERK2 causes it to form dimers, and the most flexible portions of the ERK2 molecule provide the surfaces for dimerization. It is thought that dimerization promotes nuclear localization of ERK2 by its effects on import, export or retention in cytoplasmic and nuclear compartments. Dimerization might also influence substrate interactions.

Animals↗
PubMed2000

HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1.

The serine/threonine kinase HPK1 is a member of the germinal center kinase (GCK) family that has been implicated in the regulation of MAP kinase pathways. Here, we demonstrate the involvement of HPK1 in antigen receptor signaling. Engagement of the TCR or the BCR resulted in a marked induction of HPK1 catalytic activity. Subsequent analysis revealed that Src and Syk/ZAP-70 tyrosine kinases and the adaptor proteins LAT, SLP-76, BLNK, Grb2, and Grap are involved in HPK1 activation. Overexpression of HPK1 inhibited TCR activation of AP-1 and ERK2, whereas the kinase-inactive mutant of HPK1 potentiated these responses. Neither form of HPK1 affected PMA or v-Ras activation of AP-1 and ERK2. Thus, HPK1 is a negative regulator of the TCR-induced AP-1 response pathway.

Adaptor Proteins, Signal Transducing↗