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 kinas e (MAPK) from Xenopus laevis has been studied to detect the conformati onal changes in the region of regulatory phosphorylation upon enzyme a ctivation, Slow autophosphorylation of Xenopus MAPK occured predominan tly on tyrosine, the major phosphoregulatory site of MAPKs, through an intramolecular mechanism and was accompanied by a low magnitude stimu lation 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 prev iously reported reversibility of many tyrosine kinase reactions, the t yrosine phosphorylation of Xenopus MAPK was found to be irreversible i n the presence of high ADP concentrations, although ADP could competit ively inhibit both autophosphorylation and myelin basic protein phosph orylation. We concluded, therefore, that the phosphoregulatory tyrosin e is no more accessible to an intramolecular phosphotransferase reacti on and is out of the reach of the enzyme catalytic center after phosph orylation. The conformational changes in the region of regulatory phos phorylation resulted in a reduced immunoprecipitation of autophosphory lated and MAPK-kinase-phosphorylated forms of the enzyme by a polyclon al antibody raised against a synthetic peptide corresponding to residu es 173-197 of Xenopus MAPK which includes the sites of regulatory phos phorylation. The reduced recognition was not clue to the phosphorylati on itself, since the antibody efficiently immunoprecipitated SDS-denat ured forms of the phosphorylated enzyme. The antibody was not a neutra lizing antibody, allowing unphosphorylated MAPK to undergo autophospho rylation while in the immune complex. However, autophosphorylation cau sed a release of phosphorylated enzyme from the immune complex, sugges ting that dramatic conformational changes, which could even overcome t he antibody constraints, took place in the phosphoregulatory region of MAPK upon enzyme activation.