Ordered phosphorylation of p42mapk by MAP kinase kinase

Abstract

Preparation of milligram amounts of [³²P]p42^mapk, phosphorylaled at Tyr¹⁸⁵ or diphosphorylated at Tyr¹⁸⁵/Thr¹⁸³, for use as specific protein phosphatase substrates is described. Tyr- but not Thr-phosphorylated p42^mapk, accumulates when ATP is limiting. Furthermore, Tyr¹⁸⁵-phosphorylated p42^mapk exhibits an apparent 10-fold decrease in apparent K _m (46.6 ± 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (∼476 nM). We conclude that Tyr¹⁸⁵ precedes Tyr¹⁸⁵ phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42^mapk for MAP kinase kinase.