Use of a drug‐resistant mutant of stress‐activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580

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114

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC 50 ‫؍‬ 0.6 and 4.7 M). At higher concentrations A291077 but not A304000 inhibited JNK2␣ (IC 50 ‫؍‬ 3.5 M). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50 -60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.The p38 mitogen-activated protein kinases (p38), also referred to as stress-activated protein kinases-2, are a family of proline-directed serine/threonine kinases (1, 2). At least four isoforms, the products of different genes, have been cloned and are 60 -70% identical in their amino acid sequence. The most commonly used nomenclature of these isoforms are p38␣ (3, 4), p38␤ (5, 6), p38␥ (7,8), and p38␦ (9, 10). A splice variant of the p38␤, referred to as p38␤2, has also been described (11). Northern blot analysis has shown a wide tissue distribution of these isoforms, although p38␤ and p38␥ are preferentially expressed in skeletal muscle (5, 9). In addition to stressors, members of this family of protein kinases can also be activated by growth factors (12-15).Full activation of p38 by pro-inflammatory cytokines requires phosphorylation of Thr-180 and Tyr-182 found within a TGY tripeptide motif in the activation loop of the kinase (16). This double phosphorylation is catalyzed by the dual-specific MAPK 1 kinases MKK3 and MKK6 and possibly via auto-phosphorylation (17). It is remarkable that stimuli that increase p38 phosphorylation such as insulin-like growth factor-1 (18), muscle contraction (19 -21), lipoic acid (22), 5-aminoimidazole-4-carboxamide ribonucleoside (23), pro-inflammatory cytokines (18), protein synthesis inhibitors (24, 25), hyperosmolar stress (26), and preconditioning (ischemia/reperfusion) (27) also elevate glucose uptake. Importantly, the pyridinylimidazole inhibitor of p38, SB203580, reduced the stimulation of glucose uptake by all of the above stimuli incl...

Section: Inhibition Of P38 Kinase Activity By the Novel Compoundsmentioning

confidence: 87%

“…The p38AGF mutant has been used previously to study the functional role of p38 in cells in culture and in mice (33)(34)(35)46). Expression of p38AGF was induced after 3T3-L1 cells differentiated into adipocytes.…”

Section: Discussionmentioning

confidence: 99%

A Dominant-negative p38 MAPK Mutant and Novel Selective Inhibitors of p38 MAPK Reduce Insulin-stimulated Glucose Uptake in 3T3-L1 Adipocytes without Affecting GLUT4 Translocation

Somwar¹,

Koterski²,

Sweeney³

et al. 2002

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114

“…Recently, a chemical genetic approach first described in vitro (12,13) has been employed in mice in which Thr 106 in p38␣ was substituted with a more bulky methionine, disrupting the hydrophobic groove on which many inhibitors depend for binding (8). We have made use of these mice to address the uncertainties highlighted above.…”

mentioning

confidence: 99%

A Chemical Genetic Approach Reveals That p38α MAPK Activation by Diphosphorylation Aggravates Myocardial Infarction and Is Prevented by the Direct Binding of SB203580

Kumphune

Bassi

Jacquet

et al. 2010

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38␣ alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wildtype hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38␣ kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38␣ (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.

“…In cell types such as macrophages, which only express p38␣ and not the other SB 203580-sensitive isoform, p38␤ (44), it can also be concluded that the critical activity in the phenomenon under study was that of p38␣. Expression of an SB 203580-resistant mutant of p38 MAPK was used to demonstrate that the inhibitory effect of SB 203580 on activation of MAPKAP kinase-2 and mitogen-and stress-activated protein kinase-1 involved inhibition of p38 MAPK but that the ability of SB 203580 to activate Raf-1 did not (45).…”

mentioning

confidence: 99%

Defining the Involvement of p38α MAPK in the Production of Anti- and Proinflammatory Cytokines Using an SB 203580-resistant Form of the Kinase

Guo

Gerl

Schrader

2003

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1␤, IL-6, IL-10, and tumor necrosis factor ␣ (TNF␣) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38␣ (SBR-p38␣) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1␤, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNF␣ stimulated by ligation of the Fc-␥ receptor of the mast cell MC/9. Expression of SBR-p38␣ in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNF␣ but not of IL-10. Expression of SBR-p38␣ in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-␥ induced by co-ligation of CD3 and CD28 but not the production of interferon-␥ or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.