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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T cell signaling via the CD4 surface antigen is mediated by the associated tyrosyl protein kinase p56lck. The 42-kilodalton mitogen-activated protein (MAP) kinase (p42mapk) was tyrosyl-phosphorylated and activated after treatment of the murine T lymphoma cell line 171CD4+, which expresses CD4, with antibody to CD3. Treatment of the CD4-deficient cell line 171 with the same antibody did not result in phosphorylation or activation of p42mapk. Purified p56lck both tyrosyl-phosphorylated and stimulated the seryl-threonyl phosphotransferase activity of purified p44mpk, a MAP kinase isoform from sea star oocytes. A synthetic peptide modeled after the putative regulatory phosphorylation site in murine p42mapk (Tyr185) was phosphorylated by p56lck with a similar Vmax, but a fivefold lower Michaelis constant (Km) than a peptide containing the Tyr394 autophosphorylation site from p56lck. MAP kinases may participate in protein kinase cascades that link Src family protein-tyrosyl kinases to seryl-threonyl kinases such as those encoded by rsk and raf, which are putative substrates of MAP kinases.
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PMID:Tyrosyl phosphorylation and activation of MAP kinases by p56lck. 131 Nov 28

We previously reported that p56lck expression is upregulated in human B lymphocytes upon mitogenic stimulation. In this report, we characterized the molecules associated with p56lck in vivo in leukemic B cells costimulated with anti-mu Ab and IL-2 for 72 h. In vitro phosphorylation after p56lck immunoprecipitation indicated that p56lck is associated in vivo with the beta chain of the IL-2 receptor and p42 MAP kinase as well as a number of other proteins. Moreover, p56lck-associated MAP kinase is tyrosine and threonine phosphorylated, suggesting that it is activated. Prevention of DNA synthesis with aphidicolin abrogated this molecular association, and furthermore, cell cycle analysis with IL-2-dependent T cells showed that in cells in G1, MAP kinase was not associated to p56lck, whereas this p56lck-MAP kinase association was observed when cells are in S phase. Thus, p56lck and MAP kinase are only associated during S phase. These data suggest that MAP kinase in association with p56lck is directly involved in the control of IL-2-mediated DNA synthesis of both B and T lymphocytes.
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PMID:In vivo association between p56lck and MAP kinase during IL-2-mediated lymphocyte proliferation. 749 46

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.
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PMID:MAP kinases from bovine brain: purification and characterization. 751 82

Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500-2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the kinase domain of the epidermal growth factor receptor, the src-family protein kinases p56lck and p60c-src, myelin basic protein kinase-1, or protamine kinase. Phosphoamino acid analysis demonstrated that the kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the protamine kinase phosphorylated myelin basic protein on serines, and that myelin basic protein kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.
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PMID:Autophosphorylation-activated protein kinase inactivates the protein tyrosine phosphatase activity of protein phosphatase 2A. 752 89

Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.
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PMID:Evidence of redox-linked signaling for producing a giant signal complex. 753 34

The glycosylphosphatidylinositol (GPI)-anchored CD59 protein (human protectin) protects cells against complement-induced lysis, binds to CD2 and also transduces activation signals within T cells. We have further examined the biochemical signals transduced by CD59 and addressed its role in regard to the CD3-mediated signaling cascade. We show here that CD59 cross-linking induces a time-dependent activation of p56lck and of p70zap (ZAP-70) in CD3-positive Jurkat cells, leading to the stimulation of the T cell receptor zeta/ZAP-70 signaling cascade and interleukin-2 (IL-2) synthesis. Cross-linking of CD59 on peripheral T cells and thymocytes induces tyrosine phosphorylations identical to those seen in Jurkat cells and this is followed by lymphokine production and proliferation. In contrast, only activation of CD59-associated p56lck occurs in CD3-negative Jurkat cells, while IL-2 production is impaired, consistent with the lack of ZAP-70 tyrosine phosphorylation observed in these cells. CD59 triggers activation events even in the absence of CD3/T cell receptor expression in Jurkat cells. CD59 cross-linking synergizes with sub-optimal doses of phorbol ester for activation of the protein kinase C and of the p42mapk, as shown by in vitro phosphorylation of histone HIIIS and myelin basic protein, respectively, and leads to CD25 but not CD69 expression. In conclusion, at least two signaling pathways are triggered through CD59, the first one involving ZAP-70 activation and leading to IL-2 secretion and a second pathway observed in the absence of ZAP-70 activation leading to CD25 expression. These two pathways are likely to be involved in the modulation of T cell activation by CD59 protein.
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PMID:The glycosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor zeta/ZAP-70-dependent and -independent signaling pathways in T cells. 754 90

The very late activated Ag (VLA) molecules not only mediate T cell adhesions, but also provide costimulation in a TCR/CD3-dependent manner. However, little is known about the signals mediated by the ligation of VLA molecules. Previous work from our laboratory identified a 105-kDa protein that is predominantly phosphorylated on tyrosine residue upon engagement of VLA-4 in a human T lymphoblastic cell line, H9, and in peripheral T cells. In the present study, we have shown that the A and B epitope of VLA-4 plays a key role in VLA-4-mediated T cell costimulation. Moreover, we have demonstrated that the solid phase cross-linking of VLA-4 using Ab (against A and B) or the CS-1 region of fibronectin, stimulated tyrosine phosphorylation of 140-, 120-, 80- to 70-, 60- to 55-, 50-, and 45-kDa proteins in addition to the 105-kDa protein. In contrast, Ab ligation of the C epitope of VLA-4 mainly induced tyrosine phosphorylation of pp105, weakly induced other protein tyrosine phosphorylation, and additionally induced only minimal T cell costimulation. Using immunoblotting, we have identified some of the tyrosine-phosphorylated proteins to be phospholipase C gamma (pp140), pp125 focal adhesion kinase (pp120), paxillin (pp70 and pp50), p59fyn/p56lck (pp60-55), and mitogen-activated protein kinase (pp45). Since solid phase cross-linking of VLA-4 by B2 epitope-specific Ab induced T cell costimulation most strongly via the CD3 pathway, our results suggested that the above tyrosine-phosphorylated proteins may play an important role in VLA-4-mediated T cell costimulatory signaling events.
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PMID:Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4. 767 11

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.
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PMID:Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein. 828 30

We investigated activation of mitogen-activated protein (MAP) kinase, also known as microtubule associated protein-2 kinase (MAP-2K), by recombinant interleukin-2 (rIL-2) in phytohaemagglutinin (PHA)-induced peripheral blood lymphoblasts (PBL). MAP-kinase activation has been implicated in growth of lymphocytes and other cell types. Enzyme activity was purified from cell lysates by ion-exchange chromatography and activity measured by the ability to phosphorylate the substrates MAP-2 and myelin basic protein peptide (APRTPGGRR) in vitro. Recombinant IL-2 stimulated a variable (two-to 10-fold) and evanescent MAP-2K response which was dose dependent over the range 0-50 U/ml. In contrast to MAP-kinase activation by the CD3 receptor, activation by the IL-2 receptor (IL-2R) proceeded independently from protein kinase C (PKC) and extracellular-free Ca2+. MAP-kinase activation by CD3 involves an activation cascade which depends on Ca2+ influx and PKC activation. These events culminate in tyrosine phosphorylation and activation of MAP kinase. Recombinant IL-2 induced tyrosine phosphorylation of several intracellular proteins, including a 40,000 MW substrate which co-electrophoresed with ERK-2 on SDS-PAGE. The ERK-2 gene encodes a 41,000 MW MAP-2K and is subject to regulation by a variety of mitogens and growth factors in lymphocytes and non-lymphoid cells. MAP-kinase activation by rIL-2 was abrogated when PHA blasts were pretreated with the tyrosine protein kinase (TPK) inhibitor, methyl-2,5-dihydroxy-cinnamate. Although the TPK, p56lck, has been implicated in the activation of MAP kinase and the function of IL-2R, we found no mobility shift from a 56,000 to a 60,000 MW position as seen during PKC activation. Together these data suggest that tyrosine phosphorylation is critical to IL-2-mediated signal transduction and that MAP kinase is one of the cellular intermediates involved in this pathway.
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PMID:Activation of mitogen-activated protein kinase/ERK-2 in phytohaemagglutin in blasts by recombinant interleukin-2: contrasting features with CD3 activation. 838 29

The induction of T-cell growth by the T-cell antigen receptor (TcR) is dependent on a co-ordinated process of phosphorylation and dephosphorylation of intracellular proteins. An intermediary in this signalling pathway is the serine kinase, p42 mitogen-activated protein kinase (p42MAPK), also known as microtubule-associated protein-2 kinase (MAP-2K). MAP-kinase is activated upon the acquisition of tyrosine as well as threonine phosphate groups and removal of either by specific tyrosine or serine/threonine phosphatases abrogates kinase activity. Okadaic acid (OA), a tumour promoter and potent inhibitor of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A), induced MAP-kinase activity in Jurkat T cells in a dose-dependent fashion with optimal effect at 1 microM. Compared to rapid activation (peak < 10 min) of MAP-kinase by another tumour promoter, the phorbol ester, PMA, the effect of OA was delayed (> 30 min) and more sustained. In spite of activating a growth-promoting kinase, OA differed from PMA by its lack of mitogenic activity and failure to induce CD25 [interleukin-2R alpha (IL-2R alpha)] expression in normal human T cells. This implies that PP1 and PP2A also act downstream of MAP-kinase to facilitate later cell cycle events. PMA induced a 42,000 MW tyrosine phosphoprotein which co-electrophoresed and co-chromatographed with ERK-2, a p42 MAP-kinase. Although OA induced an identical Mono-Q peak, there was less avid tyrosine phosphorylation of p42. OA also differed from PMA to the extent by which it induced mobility shift of the tyrosine protein kinase, p56lck, which has been implicated in p42MAPK activation in T cells. Taken together, these results indicate that OA and PMA exert both overlapping as well as divergent effects on lymphocyte growth pathways.
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PMID:Contrasting effects of two tumour promoters, phorbol myristate acetate and okadaic acid, on T-cell responses and activation of p42 MAP-kinase/ERK-2. 838 30


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