The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition.     

Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.     

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.     

MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates.     

Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.     

The complete pathway for catalytic activation of the mitogen-activated protein kinase, ERK2

The mitogen-activated protein (MAP) kinase ERK2 is an essential signal transduction molecule that mediates extracellular signaling by all polypeptide growth factors. Full activation of ERK2 requires phosphorylation at both a threonine residue (Thr(183)) conserved in most protein kinases as well as a tyrosine residue (Tyr(185)) unique to members of the mitogen-activated protein kinase family. We have characterized the kinetic role of phosphorylation at each site with respect to the overall activation mechanism, providing a complete picture of the reaction steps involved. Phosphorylation at Tyr(185) serves to configure the ATP binding site, while phosphorylation at both residues is required to stabilize binding of the protein substrate, myelin basic protein. Similar control mechanisms are employed to stabilize ATP and myelin basic protein in the phosphoryl group transfer reaction, accounting for the enormous increase in turnover rate. The mechanism of ERK2 activation is kinetically similar to that of the cell cycle control protein, cdk2/cyclinA. Phosphorylation of Tyr(185) in ERK2 and association of cyclinA with cdk2 both serve to stabilize ATP binding. Subsequent phosphorylation of both enzymes on threonine serves to stabilize binding of the phosphoacceptor substrate.     

Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34cdc2.

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.     

Signal transduction within the nucleus by mitogen-activated protein kinase.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.     

Functional expression in mammalian cells of a full-length cDNA coding for the pp42/MAP kinase (p42mapk) protein.

We recently cloned from a mouse 3T3 cell cDNA library a cDNA with sequence similarity to the p42mapk protein and other members of the MAP kinase family. To determine with certainty which member of the family this clone encodes, we have expressed the cDNA in COS cells and characterized the protein product. When the pSV2MAP plasmid carrying the full-length clone was transfected into COS cells, a protein of 42,000 Da was expressed. This 42 kDa protein displayed chromatographic properties indistinguishable from the endogenous p42mapk, and could be separated from the closely related pp44. In addition, upon serum stimulation, the 42 kDa protein became tyrosine-phosphorylated and enzymatically active towards the substrate myelin basic protein. We conclude that this clone codes for a functional p42mapk protein kinase.     

MAP kinase kinase from rabbit skeletal muscle. A novel dual specificity enzyme showing homology to yeast protein kinases involved in pheromone-dependent signal transduction.

MAP kinase kinase (MAPKK) was purified 30,000-fold to homogeneity from extracts of rabbit skeletal muscle and shown to be a monomeric protein of apparent molecular mass 44 kDa. MAPKK activated the 42 kDa isoform of MAP kinase by phosphorylation of Thr-183 and Tyr-185, and phosphorylated itself slowly on tyrosine, threonine and serine residues, establishing that it is a 'dual specificity' protein kinase. Peptide sequences from MAPKK were homologous to other protein serine/threonine kinases, especially to the subfamily that includes yeast protein kinases that lie upstream of yeast MAP kinase homologues in the pheromone-dependent mating pathways.     

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.     

Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase.

p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase.     

Thrombin activates mitogen-activated protein kinase in primary astrocyte cultures

Thrombin is known to evoke numerous inflammatory and proliferative responses in a wide variety of its target cells. Recent studies have demonstrated morphoregulatory and mitogenic effects of thrombin on astroglial cells (astrocytes). The present study deals with thrombin-induced activation of mitogen-activated protein (MAP) kinase in primary cultures of rat astrocytes. Treatment of serum-starved astrocytes with thrombin resulted in a rapid activation of tyrosine (Tyr) phosphorylation of a set of proteins including a prominent one with a molecular mass of 42 kDa (p42). The identity of p42 with MAP kinase was confirmed by MAP kinase-immunoreactivity of isolated [i.e., immunoprecipitated with anti-phosphotyrosine (PY) antibodies] p42 and by increased myelin basic protein (MBP) kinase activity present in MAP kinase immunoprecipitates of thrombin-treated cultures. Pertussis toxin (PTX) pretreatment failed to inhibit thrombin stimulation of p42 phosphorylation, indicating the lack of involvement of PTX sensitive G proteins in the mechanism of activation of MAP kinase by thrombin. Chronic exposure of cultures to phorbol 12-myristate 13-acetate to down-regulate PKC resulted in an attenuation of thrombin-induced p42 Tyr phosphorylation, although H-7, a known PKC inhibitor, failed to block thrombin effect. However, staurosporine, a nonspecific protein kinase inhibitor, prevented the activation of p42 phosphorylation. It is concluded that thrombin induces MAP kinase activation in astrocytes by a mechanism involving a staurosporine-sensitive pathway. © 1995 Wiley-Liss, Inc.     

Hierarchical phosphorylation of the TNF-alpha receptor, TNF-R1, by p42Mapk/Erk at basic Pro-directed kinase sites

Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.     

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172)

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.