Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event
Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event
复制标题
DOI：
10.1042/bj20031259
复制
发表时间：
2004-01-01
影响因子：
4.1
通讯作者：
Cohen, P
中科院分区：
文献类型：
--
作者：
Cole, A;Frame, S;Cohen, P
内源性GSK 3 α的磷酸化在HEK 293或SH-SY 5 Y细胞中，通过与GSK 3的药理学抑制剂孵育，而不是通过Src家族抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3，4-d]嘧啶（PP 2）或一般蛋白酪氨酸激酶抑制剂（染料木黄酮），抑制了Tyr（279）处的糖原合成酶激酶-3 α和Tyr（216）处的GSK 3 β。转染到HEK-293细胞或大肠杆菌中的GSK 3，β在Tyr（216）处磷酸化，但无催化活性的突变体没有。GSK 3 β在昆虫Sf 21细胞或E.大肠杆菌的Tyr（216）被广泛磷酸化，但在体外与MgATP孵育后，该位置缺少磷酸的少数分子可以在Tyr（216）处自磷酸化。自磷酸化的速率不受稀释的影响，并且被GSK 3抑制剂kenpaullone抑制。野生型GSK 3不能催化缺乏Tyr（216）磷酸的催化失活的GSK 3 β的酪氨酸磷酸化。我们的研究结果表明，GSK 3的酪氨酸磷酸化是我们所研究的细胞中的分子内自磷酸化事件，并且这种修饰增强了酶的稳定性。
Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr(279) and GSK3beta at Tyr(216) was suppressed in HEK293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3,beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr(216), but catalytically inactive mutants did not. GSK3beta expressed in insect Sf21 cells or E. coli was extensively phosphorylated at Tyr(216), but the few molecules lacking phosphate at this position could autophosphorylate at Tyr(216) in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3 was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr(216). Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.