Arsenite blocks growth factor induced activation of the MAP kinase cascade, upstream of Ras and downstream of Grb2-Sos.

Pretreatment of cells with 0.5 mM sodium arsenite (but not other activators of stress-activated MAP kinase cascades) prevents the activation of p21Ras and strongly suppresses the activation of c-Raf and the MAP kinase cascade by a variety of growth factors. Arsenite appears to exert its effect by preventing the guanine nucleotide exchange factor mSos from converting Ras to its active GTP-bound state. Exposure to arsenite may be a simple way of assessing whether Ras plays an essential role in mediating activation of the MAP kinase cascade by extracellular signals.

The p38/reactivating kinase mitogen-activated protein kinase cascade mediates the activation of the transcription factor insulin upstream factor 1 and insulin gene transcription by high glucose in pancreatic beta-cells.

Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic beta-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter. Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation. In human islets, or in the mouse beta-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism. The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors. Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580. Cellular stresses that trigger the activation of SAPK2 and MAPKAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580. IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form. SAPK2 could not be replaced by p42 MAP kinase, MAPKAP kinase-2, or MAPKAP kinase-3. The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase. Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role. SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.

Identification of anisomycin-activated kinases p45 and p55 in murine cells as MAPKAP kinase-2.

We recently showed that EGF and anisomycin activate two kinases, p45 and p55, whose distinguishing feature is that their detection in in-gel kinase assays is enhanced by copolymerised poly-Glu/Tyr or poly-Glu/Phe (Cano E, Hazzalin CA and Mahadevan LC, Mol. Cell. Biol., 20:117-121). Their activation characteristics and sizes are strikingly similar to those of JNK/SAPKs, which are also strongly activated by anisomycin. However, we show here that p45 and p55 are not JNK/SAPKs but murine forms of MAPKAP kinase-2 because: (i) Detection of immunoprecipitated JNK/SAPKs is completely dependent on the presence of c-Jun as substrate in the in-gel kinase assays, whereas detection of p45 and p55 is not. (ii) Detection of p45 and p55 activity is enhanced by the presence of poly-Glu/Tyr or poly-Glu/Phe, whereas JNK/SAPKs are not detectable under these conditions. (iii) Although the sizes of the murine JNK/SAPKs and MAPKAP K-2 are similar, human JNK/SAPKs migrate at 45 and 55 kDa whereas human MAPKAP K-2 migrates at 50 kDa; the poly-Glu/Tyr-enhanced activity in human cells migrates at 50 KDa. (iv) Purified rabbit muscle MAPKAP K-2 is detectable as two bands of activity on in-gel kinase assays and their detection is enhanced by poly-Glu/Tyr. (v) Finally, the anisomycin-activated poly-Glu/Tyr-enhanced p45 and p55 kinases can be immunoprecipitated from murine cells using an anti-MAPKAP K-2 antibody. Thus, EGF- and anisomycin-activated p45 and p55 are not JNK/SAPKs but MAPKAP K-2, implying that both these agents activate the p38/RK MAP kinase cascade.

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.

Phosphotyrosine residues in the nerve-growth-factor receptor (Trk-A). Their role in the activation of inositolphospholipid metabolism and protein kinase cascades in phaeochromocytoma (PC12) cells.

PC12 cells, which lack platelet derived-growth-factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the beta-PDGF receptor and the intracellular and transmembrane domains of the nerve-growth-factor receptor Trk-A (termed PT-R). Mutation of the Trk-A residue Tyr490 to phenylalanine prevents the association with Shc, while similar mutations at Tyr751 or Tyr785 are reported to prevent interaction of Trk-A with the p85 subunit of inositol phospholipid 3-kinase and phospholipase C-gamma 1, respectively. The strong and sustained activation of p42 and p44 mitogen-activated-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffected by mutation of Tyr785 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr785. Mutation of Tyr490 reduced by 70% the PDGF-induced increase in inositol phospholipid 3-kinase activity immunoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF-induced increase in the intracellular products of inositol phospholipid 3-kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutation of Tyr490 or Tyr751) abolished PDGF-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. Mutation of Tyr490, alone or in combination with mutation of Tyr751 and Tyr785, had no effect on the PDGF-induced activation of p70 S6 kinase (p70S6K). However, the activation of p70S6K by PDGF (or nerve growth factor), but not the activation of mitogen-activated-protein kinase, was prevented by two structurally unrelated inhibitors of inositol phospholipid 3-kinase, wortmannin or LY294002. Our results demonstrate the following: (1) the phosphorylation of Tyr490 plays a major role in the activation of inositol phospholipid 3-kinase and formation of 3-phosphorylated inositol lipids and confirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C-gamma 1 in vivo. (2) Tyr490 phosphorylation (but not inositol phospholipid 3-kinase activation) is also required for strong and sustained activation of mitogen-activated-protein kinase and neuronal differentiation, while the smaller and more transient activation of mitogen-activated-protein kinase, produced by the activation of phospholipase C-gamma 1 is insufficient to trigger the neuronal differentiation of PT-R cells. (3) Inositol phospholipid 3-kinase is required for the activation of p70S6K, but only a small increase in inositol phospholipid 3-kinase activity and the level of 3-phosphorylated inositol lipids is required for maximal p70S6K activation.

SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1.

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739-746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 microM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.

Activation of the MAP kinase homologue RK requires the phosphorylation of Thr-180 and Tyr-182 and both residues are phosphorylated in chemically stressed KB cells.

A MAP kinase homologue, termed the reactivating kinase (RK), lies in a signalling pathway which mediates cellular responses to stress. Here we demonstrate that the stress-induced activation of the RK in human KB cells is accompanied by the phosphorylation of Thr-180 and Tyr-182, and that the phosphorylation of both residues is required for the activation of this enzyme.

Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.

Characterization of fly rhodopsin kinase.

Rhodopsin kinase activity of Musca domestica was characterized in a reconstitution assay, using urea-treated eye membranes as substrate and a purified fraction of eye cytosol as the enzyme. Analysis of kinase activity in fly eye, brain and abdomen extracts by reconstitution assays revealed that fly rhodopsin kinase is an eye-specific enzyme. It preferentially phosphorylates the light-activated form of rhodopsin (metarhodopsin) and has little activity with other protein substrates. Rhodopsin kinase binds to metarhodopsin and is released from rhodopsin-containing membranes. Metarhodopsin is a poor substrate for kinases from tissues other than the eye, making it a unique substrate for rhodopsin kinase. Rhodopsin kinase is inhibited by heparin, but not by the protein inhibitor of cAMP-dependent protein kinase. Its Km for ATP is 9 microM. Since fly rhodopsin is coupled to phospholipase C, studies of the interaction of rhodopsin with rhodopsin kinase can be useful in analysis of the reactions that lead to termination of the inositol-phospholipid-signaling pathway.