METABOLIC LABELING OF MITOGEN-ACTIVATED PROTEIN-KINASE KINASE IN A431CELLS DEMONSTRATES PHOSPHORYLATION ON SERINE AND THREONINE RESIDUES

Mitogen-activated protein (MAP) kinase kinase is an enzyme that activa tes the growth factor-regulated MAP kinase in vitro by a mechanism tha t involves direct phosphorylation of MAP kinase on tyrosine and threon ine residues. MAP kinase kinase is stimulated by growth factor treatme nt of cells and has been shown to be inactivated with protein phosphat ases, suggesting that it is regulated by protein phosphorylation. Anal ysis of two epidermal growth factor-stimulated forms of MAP kinase kin ase, purified from P-32-labeled A431 cells, shows that the kinase is p hosphorylated on serine and threonine residues and that treatment with protein phosphatases leads to serine dephosphorylation. Under conditi ons that lead to complete inactivation, only partial dephosphorylation of MAP kinase kinase is observed. Consistent with this finding, inact ive forms of MAP kinase kinase, which separate from active forms durin g the course of purification, are also observed to be phosphorylated i n intact cells.