Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2).
Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.
Krishnan
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Department of Oncology and Immunology, Lederle Laboratories, Pearl River, NY 10965.
Kovacs
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Sass
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D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D000409:Alanine