Thomas G, Haavik J, Cohen P
Department of Biochemistry, University of Dundee, Scotland.
Eur J Biochem. 1997 Aug 1;247(3):1180-9. doi: 10.1111/j.1432-1033.1997.01180.x.
Sodium arsenite and osmotic shock both stimulated stress-activated protein kinase-2 (SAPK2, also termed RK, p38, CSBP and Mxi2) and its downstream target mitogen-activated protein kinase (MAP kinase)-activated protein kinase-2 (MAPKAP-K2) in bovine adrenal chromaffin and rat PC12 cells. The same stimuli also increased tyrosine hydroxylase activity 2-3-fold and induced its phosphorylation at Ser19, a residue phosphorylated by MAPKAP-K2 in vitro. The arsenite-induced activation of tyrosine hydroxylase and its phosphorylation at Ser19 were prevented by SB 203580 at concentrations similar to those that inhibited SAPK2 in vitro. These results indicate that MAPKAP-K2 mediates the stress-induced activation of tyrosine hydroxylase. SB 203580 had no effect on the phosphorylation or activation of tyrosine hydroxylase induced by nerve growth factor or forskolin, which trigger the phosphorylation of Ser31 and Ser40, respectively. Stimulation of bovine adrenal chromaffin cells with acetylcholine activated SAPK2 and MAPKAP-K2, as well as p42/p44 MAP kinases and their downstream target MAPKAP-K1. The half-times for activation of MAPKAP-K1 and MAPKAP-K2 (1 min) were similar. In contrast, the activation of tyrosine hydroxylase by acetylcholine peaked within 1 min and gradually declined thereafter. Neither SB 203580 (which blocked the activation of MAPKAP-K2 by acetylcholine) nor PD 98059 (which prevented the activation of p42/p44 MAP kinases by acetylcholine) affected tyrosine hydroxylase activation after 1 min, but these compounds inhibited activation by 40-50% after 5 min. PD 98059 prevented the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Ser31, the residue targetted by p42/p44 MAP kinases in vitro, but did not inhibit the phosphorylation of Ser40 (which is phosphorylated by MAPKAP-K1 in vitro). Our results establish that p42/p44 MAP kinases mediate the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Ser31. SB 203580 did not suppress the phosphorylation of Ser19 or Ser40 by acetylcholine but, like PD 98059, this drug decreased the phosphorylation of Ser31. SAPK2 may therefore contribute to the acetylcholine-induced activation of tyrosine hydroxylase by facilitating (in an unknown way) its phosphorylation by MAP kinases.
亚砷酸钠和渗透休克均能刺激牛肾上腺嗜铬细胞和大鼠PC12细胞中的应激激活蛋白激酶2（SAPK2，也称为RK、p38、CSBP和Mxi2）及其下游靶点丝裂原活化蛋白激酶（MAP激酶）激活的蛋白激酶2（MAPKAP-K2）。相同的刺激还使酪氨酸羟化酶活性提高了2 - 3倍，并诱导其在Ser19位点磷酸化，该位点在体外可被MAPKAP-K2磷酸化。亚砷酸钠诱导的酪氨酸羟化酶激活及其在Ser19位点的磷酸化可被SB 203580阻断，其浓度与在体外抑制SAPK2的浓度相似。这些结果表明，MAPKAP-K2介导了应激诱导的酪氨酸羟化酶激活。SB 203580对神经生长因子或福斯可林诱导的酪氨酸羟化酶磷酸化或激活没有影响，神经生长因子和福斯可林分别触发Ser31和Ser40的磷酸化。用乙酰胆碱刺激牛肾上腺嗜铬细胞可激活SAPK2和MAPKAP-K2，以及p42/p44 MAP激酶及其下游靶点MAPKAP-K1。MAPKAP-K1和MAPKAP-K2激活的半衰期（1分钟）相似。相比之下，乙酰胆碱对酪氨酸羟化酶的激活在1分钟内达到峰值，随后逐渐下降。SB 203580（阻断乙酰胆碱对MAPKAP-K2的激活）和PD 98059（阻止乙酰胆碱对p42/p44 MAP激酶的激活）在1分钟后均不影响酪氨酸羟化酶的激活，但这些化合物在5分钟后可抑制40 - 50%的激活。PD 98059可阻止乙酰胆碱诱导的酪氨酸羟化酶在Ser31位点的磷酸化，Ser31位点是体外p42/p44 MAP激酶的作用靶点，但不抑制Ser40的磷酸化（Ser40在体外被MAPKAP-K1磷酸化）。我们的结果表明，p42/p44 MAP激酶介导了乙酰胆碱诱导酪氨酸羟化酶在Ser31位点磷酸化。SB 203580不抑制乙酰胆碱对Ser19或Ser40的磷酸化，但与PD 98059一样，该药物可降低Ser31的磷酸化。因此SAPK2可能通过（以未知方式）促进MAP激酶对酪氨酸羟化酶的磷酸化，从而参与乙酰胆碱诱导的酪氨酸羟化酶激活。
