Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp PCC 6803
Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp PCC 6803
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DOI：
10.1007/s00284-002-3887-2
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发表时间：
2003-04-01
影响因子：
2.6
通讯作者：
Ikeuchi, M
中科院分区：
文献类型：
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作者：
Kamei, A;Yoshihara, S;Ikeuchi, M
基于基因组序列，单细胞能动蓝藻Synechocystis sp. PCC 6803具有7个属于Pkn 2亚家族（spkA类似于spkG）的真核型蛋白激酶的推定基因。以前，SpkA被证明具有蛋白激酶活性，并且是细胞运动所必需的。在这里，研究了spkB的作用。将spkB基因在大肠杆菌中表达为带有His标签的融合蛋白，并通过Ni 2+亲和层析进行纯化。表达的SpkB的真核生物型蛋白激酶活性被证明为自身磷酸化和一般底物蛋白的磷酸化。SpkB在Mg ~（2+）和Mn ~（2+）存在下均表现出自磷酸化活性，但在Ca ~（2+）存在下不表现出自磷酸化活性。集胞藻spkB突变体的表型分析表明，spkB是细胞运动所必需的，但不是趋光性。这些结果表明，SpkB是真核类型的蛋白激酶，通过蛋白磷酸化调节细胞运动像SpkA。
On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily (spkA similar to spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni2+ affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg2+ and Mn2+, but not in Ca2+. Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.