Phosphoregulatory Tyrosine of Xenopus Mitogen‐Activated Protein Kinase is out of the Reach of the Enzyme Catalytic Center After Autophosphorylation

Abstract

Autophosphorylation of the recombinant mitogen-activated protein kinase (MAPK) from Xenopus Zaevis has been studied to detect the conformational changes in the region of regulatory phosphorylation upon enzyme activation. Slow autophosphorylation of Xenopus MAPK occured 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 constrast 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 SDSdenatured 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.

Constitutive association of JAK1 and STAT5 in pro‐B cells is dissolved by interleukin‐4‐induced tyrosine phosphorylation of both proteins

“…Bradshaw et al showed that interaction of the cytoplasmic tail of CD152, a mediator of T cell activation, with a clathrin‐associated protein is negatively regulated by tyrosine phosphorylation [25]. Various other studies employing epitope‐specific antibodies, protein binding assays and structural methods have provided evidence for a determinant influence of tyrosine phosphorylation on protein conformation [26–31].…”

Expression, phosphorylation, and mRNA‐binding of heterogeneous nuclear ribonucleoprotein K in Xenopus oocytes, eggs, and early embryos

“…1996), a Src family kinase, or GST‐MAPK. GST‐MAPK was purified from bacteria as described previously (Tokmakov et al . 1996).…”

“…In vitro phosphorylation assay of the immunoprecipitated xhnRNP K (see above) was also carried out with the use of the purified bovine brain c-Src or Fyn , a Src family kinase, or GST-MAPK. GST-MAPK was purified from bacteria as described previously (Tokmakov et al 1996). Phosphorylation reaction proceeded for 20 min at 30°C and was stopped by the addition of EDTA (for immunoprecipitation and immunoblotting) or Laemmli's SDS sample buffer (for immunoblotting).…”