A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress

Clifton, Andrew D.; Young, Peter R.; Cohen, Philip

doi:10.1016/0014-5793(96)00816-2

Cited by 134 publications

(125 citation statements)

References 22 publications

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“…The MAPKAP kinases MK2 and MK3 are highly similar in structure and function and, so far, no differences in their activation by p38 MAPK and their substrate specificity have been described [Clifton et al, 1996;Gaestel, 2006;Ronkina et al, 2007]. For MK2, a role in cell migration has been demonstrated which depends on its catalytic activity and on the integrity of its N-terminal proline-rich region [Kotlyarov et al, 2002].…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, it has been demonstrated that this proline-rich motif of MK2 is essential for trans-well migration of smooth muscle cells and fibroblast in a Boyden chamber experiment [Kotlyarov et al, 2002]. MK2 and MK3 are indistinguishable in regard to p38-dependent regulation and activation as well as substrate specificity [Clifton et al, 1996]. Both, MK2 and MK3, can rescue p38 level, Hsp25-phosphorylation, cytokine mRNA-stability, and CXCL1 production in MK2-deficient mouse embryonic fibroblasts [Ronkina et al, 2007].…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

Menon

Ronkina

Schwermann

et al. 2009

Cell Motil. Cytoskeleton

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The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection-based real-time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large-scale screening studies. A live cell staining lipophilic tracer-1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethyl indotricarbocyanine iodide (DiR)-is used for accurate imaging of wound closure in a simple 96-well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images are analyzed using Image J software and the migration index is calculated. Effect of cell number, time after scratch and software settings are analyzed. The method is validated by showing concentration-and time-dependent effects of cytochalasin-D on fibroblast migration. Using this assay, we quantitatively evaluate the role of the MAPK-activated protein kinases MK2 and MK3 in fibroblast migration. First, the migratory phenotype of MK2-deficient MEFs is analyzed in a retroviral rescue model. In addition, migration of MK2/3-double-deficient cells is determined and the ability of MK3 to rescue cell migration in MK2/3-double-deficient fibroblasts is demonstrated.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

Menon

Ronkina

Schwermann

et al. 2009

Cell Motil. Cytoskeleton

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“…Epidermal growth factor (EGF) was purchased from Boehringer Mannheim and the monoclonal anti-H-Ras antibody (Y13 259) from Oncogene Science Products. The antibodies against c-Raf ( Alessi et al, 1995b), MAPKAP kinase-2 (Clifton et al, 1996), MAP kinase kinase-1 (MKK1) and MAPK2 (also called ERK2) were raised in sheep at the Scottish Antibody Production Unit (Carluke, Lanarkshire, UK). The anti-MKK1 antibodies were raised against the peptide PKKKPTPIQLNPAPDG correspond-ing to residues 2 ± 17 and the anti-MAPK2 antibodies against the C-terminal peptide ± EETARFQPGYRS.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation of protein kinases c-Raf was immunoprecipitated from 400 mg cell lysate protein, MKK1 from 100 mg of cell lysate and MAPK2 and MAPKAP kinase-2 from 50 mg cell lysate protein using 5 ml of protein GSepharose conjugated to 4 mg of the appropriate antibody (Alessi et al, 1995b;Clifton et al, 1996).…”

Section: Cell Culture and Stimulationmentioning

confidence: 99%

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Effect of SB 203580 on the activity of c-Raf in vitro and in vivo

et al. 1999

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The inhibition of SAPK2a/p38 (a mitogen activated protein (MAP) kinase family member) by SB 203580 depends on the presence of threonine at residue 106. Nearly all other protein kinases are insensitive to this drug because a more bulky residue occupies this site (Eyers et al., 1998). Raf is one of the few protein kinases that possesses threonine at this position, and we show that SB 203580 inhibits c-Raf with an IC 50 of 2 mM in vitro. However, SB 203580 does not suppress either growth factor or phorbol ester-induced activation of the classical MAP kinase cascade in mammalian cells. One of the reasons for this is that SB 203580 also triggers a remarkable activation of c-Raf in vivo (when measured in the absence of the drug). The SB 203580-induced activation of c-Raf occurs without any increase in the GTP-loading of Ras, is not prevented by inhibitors of the MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this drug to SAPK2a/p38. The paradoxical activation of cRaf by SB 203580 (and by another structurally unrelated c-Raf inhibitor) suggests that inhibitors of the kinase activity of c-Raf may not be e ective as anti-cancer drugs.

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MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

Sato

1999

J. Cell. Physiol.

199

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Although the significance of vascular endothelial growth factor (VEGF) and its receptors in angiogenesis is well established, the signal transduction cascades activated by VEGF and their involvement in mediating the mitogenic response of endothelial cells to VEGF are incompletely characterized. Here we demonstrate that VEGF activates mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and p70 S6 kinase in human umbilical vein endothelial cells (HUVEC). The activation of these enzymes was assayed by kinase phosphorylation and by kinase activity towards substrates. Studies with PI 3-kinase inhibitors revealed that activation of p70 S6 kinase was mediated by PI 3-kinase. Selective inhibition of ERK, PI 3-kinase, and p70 S6 kinase with the inhibitors PD098059, LY294002, and rapamycin, respectively, inhibited VEGF-stimulated HUVEC proliferation. In marked contrast, the p38 MAP kinase inhibitor SB203580 not only failed to inhibit but actually enhanced HUVEC proliferation; this effect was associated with the phosphorylation of Rb protein. Rb phosphorylation resulted from a decrease in the level of the cdk inhibitor p27KiP1. These results indicate that the activities of ERK, PI 3-kinase, and p70 S6 kinase are essential for VEGF-induced HUVEC proliferation. p38 MAP kinase suppresses endothelial cell proliferation by regulating cell-cycle progression.

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A comparison of the substrate specificity of MAPKAP kinase‐2 and MAPKAP kinase‐3 and their activation by cytokines and cellular stress

Cited by 134 publications

References 22 publications

Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

Fluorescence‐based quantitative scratch wound healing assay demonstrating the role of MAPKAPK‐2/3 in fibroblast migration

Effect of SB 203580 on the activity of c-Raf in vitro and in vivo

MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

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