Growth factor-induced activation of a kinase activity which causes regulatory phosphorylation of p42/microtubule-associated protein kinase.

Abstract

p42/microtubule-associated protein kinase (p42 aPk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42maPk on the regulatory tyrosine And to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42maPk. This p421aPk-activating activity is capable of phosphorylating a kinase-defective p42 lPk mutant, thus confirming its activity as a kinase.p42/microtubule-associated protein (MAP) kinase (p42maPk) is a serine/threonine protein kinase which rapidly becomes phosphorylated on threonine and tyrosine (42) in quiescent cells stimulated with various agonists, resulting in enzymatic activation. Both the threonine and tyrosine phosphorylations are necessary for full enzymatic activation (7,27,44), and the phosphorylation sites have been identified as T-183 and Y-185 (35).p42maPk is the paradigmatic member of an enzyme family which is activated by agonist stimulation in various cell types and species (for a recent review, see reference 12). Members of this family have also been termed MAP-2 kinases (23,27,(40)(41)(42), myelin basic protein (MBP) kinases (2, 4, 44) or extracellularly regulated kinases (ERKs) (9, 10). The p42maPk studied here corresponds to the MAP-2 kinase originally described by Ray and Sturgill (40), the MBP kinase 1 of Krebs et al. (2,4,44), the ERK2 of Boulton et al. (9,10), and the tyrosine phosphorylated protein pp42 described earlier by Cooper and Hunter (13,24) and by our (8,42) and other laboratories (17,26).Several candidate substrates for p42maPk and its family members have been identified, including MAP-2 (40), rsk ribosomal S6 kinase (11, 47), raf protein kinase (6), epidermal growth factor (EGF) receptor (35) Any one or a combination of three general mechanisms could be responsible for the regulatory phosphorylation of p42maPk: (i) a kinase cascade, in which an upstream kinase is regulated by agonists and phosphorylates p42m Pk, much as p42maPk phosphorylates rsk kinase (47); (ii) an intramolecular autokinase reaction, in which a nonkinase cellular factor regulates the ability of p42maPk to phosphorylate itself; (iii) an intermolecular autokinase reaction, in which p42maPk molecules are activated allosterically and then engage in mutual transphosphorylations, as occurs with many tyrosine kinase receptors.Previous work has demonstrated that p42"aPk is capable of slow intramolecular autophosphorylation (14, 46, 51) on tyrosine 185 (51), the site of in vivo regulatory phosphorylation on tyrosine. This reaction is accompanied by partial enzymatic activation. Because p42maPk is capable of this basal, regulatory autophosphorylation, we and others have speculated that the autokinase activity might be exploited in the regulatory phosphorylation which accom...

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