MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.     

Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding

A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.     

WNK3 is a putative chloride-sensing kinase

The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation

Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of beta-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1-/- mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1-/- cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.     

MEK kinase 3 directly activates MKK6 and MKK7, specific activators of the p38 and c-Jun NH2-terminal kinases.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. We have examined the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38alpha were also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homologue of MEKK3, also activated MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activated recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3 or MEKK2 activates p38 in intact cells using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhanced MAPKAPK2 phosphorylation, whereas MEKK2 was less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress was abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells.     

Hepatic epidermal growth factor-regulated mitogen-activated protein kinase kinase kinase activity in the rat: lack of identity with known forms of raf and MEKK

Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.     

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.     

Conserved docking site is essential for activation of mammalian MAP kinase kinases by specific MAP kinase kinase kinases

Mammalian mitogen-activated protein kinase (MAPK) cascades control various cellular events, ranging from cell growth to apoptosis, in response to external stimuli. A conserved docking site, termed DVD, is found in the mammalian MAP kinase kinases (MAPKKs) belonging to the three major subfamilies, namely MEK1, MKK4/7, and MKK3/6. The DVD sites bind to their specific upstream MAP kinase kinase kinases (MAPKKKs), including MTK1 (MEKK4), ASK1, TAK1, TAO2, MEKK1, and Raf-1. DVD site is a stretch of about 20 amino acids immediately on the C-terminal side of the MAPKK catalytic domain. Mutations in the DVD site strongly inhibited MAPKKs from binding to, and being activated by, their specific MAPKKKs, both in vitro and in vivo. DVD site mutants could not be activated by various external stimuli in vivo. Synthetic DVD oligopeptides inhibited specific MAPKK activation, both in vitro and in vivo, demonstrating the critical importance of the DVD docking in MAPK signaling.     

A subdomain of MEKK1 that is critical for binding to MKK4

Mitogen-activated protein kinase (MAPK) cascades are central components of signal transduction pathways induced by mitogens and stresses. They consist of a three-kinase module in which a mitogen-activated protein kinase kinase kinase (MAP3K) activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates MAPK. The molecular determinants that underlie specific MAP3K-MAP2K interactions are poorly understood. In this study, we examined the interaction between the MAP3K MEKK1 and MKK4, a MAP2K of the JNK pathway. Select point mutations in subdomain X of the catalytic domain of MEKK1 (MEKK1delta) were found to impair the ability of MEKK1delta to bind to and activate MKK4. Such mutations were also found to impair MEKK1delta-induced activation of an AP1 reporter gene. These studies point to a critical role for subdomain X in the interaction of MEKK1 with MKK4.     

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis

In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding-leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses.