A strategy to make constitutively active MAP kinase by fusing with constitutively active MAP kinase kinase.
Classical mitogen-activated protein kinases (MAPKs) play a pivotal role in a variety of cellular signal transduction pathways. MAPKs are activated by phosphorylation at specific threonine and tyrosine residues catalyzed by upstream MAPK kinases (MAPKKs). Mutations of these two activation phosphorylation sites into acidic amino acids, however, do not convert MAPKs into constitutively active forms. Here, we report an approach to make a molecule with constitutive MAPK activity. The nuclear export signal-disrupted, constitutively active MAPKK was fused to the N-terminal end of wild-type MAPK. When the resulting fusion protein was expressed in Escherichia coli, the MAPK moiety became phosphorylated and the fusion protein was constitutively active as MAPK. Moreover, when expressed in mammalian cultured cells, the fusion protein was also activated as MAPK and was able to induce marked morphological changes in NIH-3T3 cells. These results suggest that the fusion protein can work as constitutively active MAPK and that this approach may be applicable to other members of the MAPK family to make constitutively active forms.
Miyata
Y
Y
Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.
Adachi
S
S
Mizuno
H
H
Nishida
E
E
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D048369:MAP Kinase Kinase 1; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases