Purcarea Cristina, Fernando Roshini, Evans Hedeel Guy, Evans David R
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 E. Canfield Street, Detroit, MI 48201, USA.
Mol Cell Biochem. 2008 Apr;311(1-2):199-213. doi: 10.1007/s11010-008-9710-z. Epub 2008 Feb 13.
Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl2 and CoCl2 but not MgCl2. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.
丝氨酸/苏氨酸激酶参与真核生物、原核生物和古生菌中复杂的相互作用信号通路。虽然大多数生物含有许多不同的激酶，但极端嗜热菌嗜热栖热菌仅编码一种假定的丝氨酸/苏氨酸激酶。一个与真核蛋白磷酸酶同源的基因与激酶基因仅相差一个碱基对。假定的激酶AaSTPK和磷酸酶AaPPM被克隆并在大肠杆菌中表达，纯化至同质且发现具有功能。AaSTPK是一种34 kDa的单体，可使用MgATP、MnATP或MnGTP作为共底物，不过MgATP似乎是首选底物。AaSTPK在一个苏氨酸残基上进行自身磷酸化，并被AaPPM去磷酸化。AaPPM磷酸酶与丝氨酸/苏氨酸磷酸酶的PPM亚家族同源，受MnCl2和CoCl2刺激，但不受MgCl2刺激。AaSTPK还使氨甲酰磷酸合成酶CPS.A亚基上的一个苏氨酸残基磷酸化。用磷酸化的CPS.A重构的氨甲酰磷酸合成酶催化活性未改变，但UMP的变构抑制和精氨酸中间体鸟氨酸的激活均明显减弱。变构调节的这些变化预计会通过解除UMP对氨甲酰磷酸合成酶活性的限制以及解开嘧啶和精氨酸生物合成的调节来激活嘧啶生物合成。CPS.A也被AaPPM去磷酸化。嗜热栖热菌位于原核生物系统发育树的最底层分支。苏氨酸/丝氨酸激酶、其同源磷酸酶和一种蛋白质底物可能是一个简单信号通路的组成部分，也许是迄今为止描述的这种调节模式的最原始例子。
