Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen‐activated protein (MAP) kinase and MAP‐kinase‐activated kinases 1 and 2

Abstract

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 3 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in v i m at comparable rates to other proteins thought to be physiological substrates of 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 moVmol subunit and 2 mol/ mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-3 ) and Serl9 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-11, which phosphorylates Serl9 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser4O about twice as fast as Serl9. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-I1 correlated with the extent 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 + 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/ mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.Tyrosine hydroxylase (TH) is a homotetrameric enzyme which catalyses the rate-limiting step in catecholamine synthesis. It is found predominantly in the adrenal medulla and central and sympathetic nervous systems, where its activity is increased by agonists that stimulate catecholamine secretion, providing a mechanism for replenishing the stores of catecholamine hormones and neurotransmitters that have been lost via secretion.

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…CaM-KII also phosphorylates TH on Ser19 (Campbell et al 1986;Isobe et al 1991). In contrast to Ser40, phosphorylation of Ser19 does not activate the enzyme directly in vitro or in vivo (Sutherland et al 1993;Haycock et al 1998). Although CaM-KII phosphorylates TH on both sites, there are discrepancies in the literature regarding the mechanism of TH activation by CaM-KII.…”

“…The tryptic digests were either subjected to highperformance liquid chromatography (HPLC) analysis immediately or stored at ) 20°C for later use (cf. Sutherland et al 1993).…”

“…Although CaM-KII phosphorylates TH on both sites, there are discrepancies in the literature regarding the mechanism of TH activation by CaM-KII. While TH from rat pheochromocytoma (PC12) cells was found to require the presence of 14-3-3 proteins to be activated (Yamauchi et al 1981;Ichimura et al 1987), we previously reported a CaM-KII activation of human TH in a Ser40-dependent manner, independent of the presence of 14-3-3 proteins (Sutherland et al 1993). TH is also activated by MAPK-activated protein kinase 2 (MAPKAP-K2), a downstream target of stressactivated protein kinase 2 (SAPK2/p38).…”

“…Ser19, Ser31 and Ser40, by a variety of protein kinases, indicating that its regulation may involve multiple signalling pathways. Studies on TH from several species suggest that Ser40 is the main site involved in direct activation of TH (Sutherland et al 1993;Lew et al 1999;Salvatore et al 2000). This serine residue is phosphorylated in vitro by cAMP-dependent kinase (PKA), protein kinase C (PKC), mitogen-activated protein kinase-activated protein kinase 1 (MAPKAP-K1) and Ca 2+ /calmodulin-dependent protein kinase II (CaM-KII; Kappock and Caradonna 1996).…”

“…TH is also activated by MAPK-activated protein kinase 2 (MAPKAP-K2), a downstream target of stressactivated protein kinase 2 (SAPK2/p38). MAPKAP-K2 phosphorylates TH on Ser19 and Ser40 in vitro in response to stresses that activate MAPKAP-K2 (Sutherland et al 1993; Thomas et al 1997), resulting in short-term activation.…”