Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf.
We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.
Moodie
S A
SA
Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195.
Paris
M J
MJ
Kolch
W
W
Wolfman
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism*; D066298:In Vitro Techniques; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D006165:Guanylyl Imidodiphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras)