PHOSPHORYLATION AND ACTIVATION OF HUMAN TYROSINE-HYDROXYLASE IN-VITROBY MITOGEN-ACTIVATED PROTEIN (MAP) KINASE AND MAP-KINASE-ACTIVATED KINASE-1 AND KINASE-2

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinase s 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphoryl ate bacterially expressed human tyrosine hydroxylase in vitro at compa rable rates to other proteins thought to be physiological substrates o f these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and - 2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, r espectively; the sites of phosphorylation were identified as Ser40 (MA PKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to c almodulin-dependent protein kinase-II, which phosphorylates Ser19 fast er than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fas t as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP k inase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the exten t of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase , but at markedly different rates (3=4 > 1 much greater than 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kin ase doubling the activity, while phosphorylation of form 1 by MAP kina se to 0.4 mol/mol subunit increased activity by 40%. The effect on act ivity of phosphorylating both Ser31 and Ser40 was not additive. The po ssible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.