Phosphoregulatory tyrosine of Xenopus mitogen-activated protein kinase is out of the reach of the enzyme catalytic center after autophosphorylation. Biochemical evidence for conformational changes upon phosphorylation.
Autophosphorylation of the recombinant mitogen-activated protein kinase (MAPK) from Xenopus laevis has been studied to detect the conformational changes in the region of regulatory phosphorylation upon enzyme activation. Slow autophosphorylation of Xenopus MAPK occurred predominantly on tyrosine, the major phosphoregulatory site of MAPKs, through an intramolecular mechanism and was accompanied by a low magnitude stimulation of the catalytic activity towards an exogenous substrate, myelin basic protein. Autophosphorylated but not unphosphorylated enzyme was shown to interact with the protein substrate. In contrast to the previously reported reversibility of many tyrosine kinase reactions, the tyrosine phosphorylation of Xenopus MAPK was found to be irreversible in the presence of high ADP concentrations, although ADP could competitively inhibit both autophosphorylation and myelin basic protein phosphorylation. We concluded, therefore, that the phosphoregulatory tyrosine is no more accessible to an intramolecular phosphotransferase reaction and is out of the reach of the enzyme catalytic center after phosphorylation. The conformational changes in the region of regulatory phosphorylation resulted in a reduced immunoprecipitation of autophosphorylated and MAPK-kinase-phosphorylated forms of the enzyme by a polyclonal antibody raised against a synthetic peptide corresponding to residues 173-197 of Xenopus MAPK which includes the sites of regulatory phosphorylation. The reduced recognition was not due to the phosphorylation itself, since the antibody efficiently immunoprecipitated SDS-denatured forms of the phosphorylated enzyme. The antibody was not a neutralizing antibody, allowing unphosphorylated MAPK to undergo autophosphorylation while in the immune complex. However, autophosphorylation caused a release of phosphorylated enzyme from the immune complex, suggesting that dramatic conformational changes, which could even overcome the antibody constraints, took place in the phosphoregulatory region of MAPK upon enzyme activation.
Graduate School of Science and Technology, Kobe University, Japan.
A A
AA
Tokmakov
S
S
Sahara
K
K
Sato
E
E
Nishida
Y
Y
Fukami
D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D014982:Xenopus laevis
D000906:Antibodies; D011994:Recombinant Proteins; D014443:Tyrosine; D017871:Calcium-Calmodulin-Dependent Protein Kinases