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
Aa. Tokmakov et al., 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, European journal of biochemistry, 241(2), 1996, pp. 322-329
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.