Prevention of MKK6-dependent activation by binding to p38alpha MAP kinase

Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.     

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4. In addition, Galpha(q) and Gbetagamma stimulated MKK3 and MKK6 activities. The MKK3 and MKK6 activations by Galpha(q), but not by Gbetagamma, were dependent on phospholipase C and c-Src. Galpha(q) stimulated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner. On the other hand, Gbetagamma activated MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-, Rac-, and Cdc42-dependent manner. Gbetagamma-induced MKK3 and MKK6 activations were dependent on a tyrosine kinase other than c-Src. These results suggest that Galpha(q) and Gbetagamma stimulate the activity of p38 MAPK by regulating MKK3 and MKK6 through parallel signaling pathways.     

Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.     

Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer

The p38 signaling pathway is activated in response to cell stress and induces production of proinflammatory cytokines. P38alpha is phosphorylated and activated in response to cell stress by MKK3 and MKK6 and in turn phosphorylates a number of substrates, including MAPKAP kinase 2 (MK2). We have determined the crystal structure of the unphosphorylated p38alpha-MK2 heterodimer. The C-terminal regulatory domain of MK2 binds in the docking groove of p38alpha, and the ATP-binding sites of both kinases are at the heterodimer interface. The conformation suggests an extra mechanism in addition to the regulation of the p38alpha and MK2 phosphorylation states that prevents phosphorylation of substrates in the absence of cell stress. Addition of constitutively active MKK6-DD results in rapid phosphorylation of the p38alpha-MK2 heterodimer.     

Dominant-negative activity of an alpha(1B)-adrenergic receptor signal-inactivating point mutation

alpha(1)-adrenergic receptors (alpha(1)-ARs) are members of the G-protein-coupled receptor (GPCR) superfamily and activate inositol phosphate (IP) turnover. We show that glycine and asparagine mutations of Phe303 in transmembrane segment VI (TMVI) of the alpha(1B)-AR, a highly conserved residue in GPCRs, although increasing agonist affinity, abolish agonist-activated IP signalling. Co-expression of the Phe303 mutants also inhibited (-)epinephrine-stimulated IP signalling by wild-type alpha(1B)-AR and other G(q)-coupled receptors, as well as IP signalling mediated by AlF(4)(-) stimulation of both wild-type G(q alpha) and a constitutively active mutant. The inability of the Phe303 mutants to signal is due to induction of a receptor conformation that dissociates G-protein binding from activation. As a result, the Phe303 mutants sequester G(q alpha) and stoichiometrically inhibit Gq signalling in a dominant-negative manner. We further show that both the enhanced basal and agonist-stimulated IP-signalling activity of the constitutively active alpha(1B)-AR mutants, C128F and A293E, are inhibited in the double mutants, C128F/F303G and A293E/F303G. Phe303, therefore, appears to be critically involved in coupling TMVI alpha-helical movement, a key step in receptor activation, to activation of the cognate G-protein.     

G(i)-dependent activation of c-Jun N-terminal kinase in human embryonal kidney 293 cells

Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether alpha subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Galpha(i1), Galpha(i2), and Galpha(i3) increased JNK activity. In contrast, constitutively activated Galpha(o) and Galpha(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Galpha(i), kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Galpha(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Galpha(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Galpha(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Galpha(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.     

Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase

p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38alpha, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38alpha correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38alpha(-/-) knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38alpha-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38alpha(-/-) cells. Pharmacological inhibition of p38alpha also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38alpha into p38alpha(-/-) cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38alpha(-/-) cells and that the 3'untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38alpha-dependent manner. Our data indicate that p38alpha can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.     

p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression

Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor beta-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38alpha by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b-->p38alpha blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.     

Mitochondrial ATP synthase residue betaarginine-408, which interacts with the inhibitory site of regulatory protein IF1, is essential for the function of the enzyme

Mitochondrial ATP synthase (F(1)F(0)-ATPase) is regulated by an intrinsic ATPase inhibitor protein, IF(1). We previously found that six residues of the yeast IF(1) (Phe17, Arg20, Glu21, Arg22, Glu25, and Phe28) form an ATPase inhibitory site [Ichikawa, N. and Ogura, C. (2003) J. Bioenerg. Biomembr. 35, 399-407]. In the crystal structure of the F(1)/IF(1) complex [Cabezón, E. et al. (2003) Nat. Struct. Biol. 10, 744-750], the core residues of the inhibitory site interact with Arg408, Arg412 and Glu454 of the beta-subunit of F(1). In the present study, we examined the roles of the three beta residues by means of site-directed mutagenesis. A total of six yeast mutants were constructed: R408I, R408T, R412I, R412T, E454Q, and E454V. The betaArg412 and betaGlu454 mutants (R412I, R412T, E454Q, and E454V) could grow on a nonfermentable lactate medium, but the betaArg408 mutants (R408I and R408T) could not. The ATPase activity of isolated mitochondria was decreased in R412I, R412T, E454Q, and E454V mutant cells, and undetectable in R408I and R408T cells. The subunits of F(1) (alpha, beta, and gamma) were detected in mitochondria from each mutant on immunoblotting, and the F(1)F(0) complex was isolated from them. These results indicate that betaArg408 is essential not for assembly of the F(1)F(0) complex but for the catalytic activity of the enzyme. In the crystal structure of F(1), betaArg408 binds to alphaGlu399 in the alpha(DP)/beta(DP) pair and seems to be important for formation of the closed alpha(DP)/beta(DP) conformation. IF(1) seems to disrupt this alpha(DP)Glu399/beta(DP)Arg408 interaction by binding to beta(DP)Arg408, and to interfere with the change from the open alpha(DP)/beta(DP) conformation to the closed conformation that is required for catalysis by F(1)F(0)-ATPase.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Computational sampling of a cryptic drug binding site in a protein receptor: explicit solvent molecular dynamics and inhibitor docking to p38 MAP kinase

An increasing number of structural studies reveal alternative binding sites in protein receptors that become apparent only when an inhibitor binds, and correct prediction of these situations presents a significant challenge to computer-aided drug design efforts. A striking example is provided by recent crystal structures of the p38 MAP kinase, where a 10A movement of the Phe169 side-chain creates a new binding site adjacent to the ATP binding site that is exploited by the diaryl urea inhibitor BIRB796. Here, we show that this binding site can be successfully and repeatedly identified in explicit-solvent molecular dynamics (MD) simulations of the protein that begin from an unliganded p38 crystal structure. Ligand-docking calculations performed on 5000 different structural snapshots generated during MD indicate that the conformations sampled are often surprisingly competent to bind the inhibitor BIRB796 in the crystallographically correct position and with docked energies that are generally more favorable than those of other positions. Similar docking studies with an ATP-binding site-directed inhibitor suggest that it may be possible to develop hybrid inhibitors that target both the ATP and cryptic binding sites simultaneously. Intriguingly, both inhibitors are occasionally found to dock correctly even with p38's "DFG" motif in the "wrong" conformation and BIRB796 can successfully dock, albeit infrequently, without significant displacement of the Phe169 side-chain; this suggests that the inhibitor might facilitate the latter's conformational change. Finally, two quite different conformations of p38's DFG motif are also sampled for extended periods of time during the simulations; these may provide new opportunities for inhibitor development. The MD simulations reported here, which total 390 ns in length, therefore demonstrate that existing computational methods may be of surprising utility in predicting cryptic binding sites in protein receptors prior to their experimental discovery.     

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.     

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.     

Structure and conformation of peptides containing the sulfonamide junction. II. Synthesis and conformation of cyclo[-MeTau-Phe-DPro-]

By applying the method of amino-acyl incorporation to sulfonamido peptides, cyclo(-MeTau-Phe-DPro-) 3 has been synthesized in high yield starting from Z-MeTau-Phe-Pro-OH. The crystal structure and the molecular conformation of 3 have been determined. Crystals are orthorhombic, s.g. P2(1)2(1)2(1), with a = 5.454, b = 13.486, c = 24.025 A. The structure has been solved by direct methods and refined to R = 0.039 for 1974 reflections with I greater than 1.5 sigma (I). The 10-measured cyclopeptide adopts a backbone conformation in the crystals characterized by Phe-DPro and DPro-MeTau peptide bonds in trans and cis conformation, respectively. Both the peptide bonds deviate significantly from planarity and the corresponding [delta omega[ values are ca. 12 degrees. The sulfonamide SO2NH junction adopts a cisoidal conformation with a C alpha 1-S1-N2-C alpha 2 torsion angle of 70.8 degrees. 13C n.m.r. data show that the trans geometry at the Phe-DPro junction found in the crystals is retained in DMSO solution. The 10-membered ring of 3 is characterized by a pseudo mirror-plane passing through the Phe nitrogen and the DPro carbonylic carbon. The DPro ring adopts a half-chair conformation. The Phe side chain conformation corresponds to the statistically most favored g- rotamer (chi 1 = -68.6 degrees). The crystal packing is characterized by a weak intermolecular hydrogen bond between NH group and the MeTau O1' oxygen.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Rac1-MKK3-p38-MAPKAPK2 pathway promotes urokinase plasminogen activator mRNA stability in invasive breast cancer cells

We reported previously that down-regulating or functionally blocking alphav integrins inhibits endogenous p38 mitogen-activated protein kinase (MAPK) activity and urokinase plasminogen activator (uPA) expression in invasive MDA-MB-231 breast cancer cells whereas engaging alphav integrins with vitronectin activates p38 MAPK and up-regulates uPA expression (Chen, J., Baskerville, C., Han, Q., Pan, Z., and Huang, S. (2001) J. Biol. Chem. 276, 47901-47905). Currently, it is not clear what upstream and downstream signaling molecules of p38 MAPK mediate alphav integrin-mediated uPA up-regulation. In the present study, we found that alphav integrin ligation activated small GTPase Rac1 preferentially, and dominant negative Rac1 inhibited alphav integrin-mediated p38 MAPK activation. Using constitutively active MAPK kinases, we found that both constitutively active MKK3 and MKK6 mutants were able to activate p38 MAPK and up-regulate uPA expression, but only dominant negative MKK3 blocked alphav integrin-mediated p38 MAPK activation and uPA up-regulation. These results suggest that MKK3, rather than MKK6, mediates alphav integrin-induced p38 MAPK activation. Among the potential downstream effectors of p38 MAPK, we found that only MAPK-activated protein kinase 2 affects alphav integrin-mediated uPA up-regulation significantly. Finally, using beta-globin reporter gene constructs containing uPA mRNA 3'-untranslated region (UTR) and adenosine/uridine-rich elements-deleted 3'-UTR, we demonstrated that p38 MAPK/MAPK-activated protein kinase 2 signaling pathway regulated uPA mRNA stability through a mechanism involving the adenosine/uridine-rich elements sequence in 3'-UTR of uPA mRNA.     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

Active mutants of the human p38alpha mitogen-activated protein kinase

Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.