Mek1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells.
MAP (mitogen-activated protein) kinases are activated by a family of dual specificity kinases called Meks (MAP kinase/Erk kinase). Mek1 can be activated by Raf by phosphorylation on serine 218 and serine 222. Mutation of these sites to acidic residues leads to constitutively active Mek1 in some cases. When fibroblast lines were infected with high titer retroviral stocks carrying these Mek1 genes, the resultant transformation and morphological changes correlated with the kinase activity of the respective Mek1 enzymes. Although [Asp218]- and [Asp218,Asp222]Mek immunoprecipitated from clonal cell lines could phosphorylate kinase-inactive Erk1 equally well in vitro, the endogenous MAP kinase activity was 5-7-fold greater in [Asp218]Mek1-infected clonal lines, and did not correlate with the degree of transformation. Analysis of the Erk1 pathway revealed Raf-1 activation, which correlated qualitatively with the MAP kinase activity seen in the [Asp218]- and [Asp218,Asp222]Mek1-infected clonal cell lines. Expression of dominant negative Ras did not affect the elevated Raf-1 activity observed in these cells, however. These data suggest that Mek1 phosphorylation site mutants activate Raf-1 and MAP kinase by a Ras-independent pathway and that the mechanism by which transformation occurs may utilize pathways that are MAP kinase-independent.
Alessandrini
A
A
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Greulich
H
H
Huang
W
W
Erikson
R L
RL
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation; D017874:Immediate-Early Proteins / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015183:Restriction Mapping
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein 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; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D017027:Protein Tyrosine Phosphatases