Biochemical and functional characterization of a eukaryotic-type protein kinase, SpkB, in the cyanobacterium, Synechocystis sp. PCC 6803.
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 approximately 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 Ni(2+) 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 Mg(2+) and Mn(2+), but not in Ca(2+). 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.
Kamei
Ayako
A
Department of Life Sciences (Biology), The University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan. kamei@rtc.riken.go.jp
Yoshihara
Shizue
S
Yuasa
Takashi
T
Geng
Xiaoxing
X
Ikeuchi
Masahiko
M
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000502:physiology; D005057:Eukaryotic Cells / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D008670:Metals / Q000378:metabolism; D018524:Phototropism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology
D001426:Bacterial Proteins; D008670:Metals; D011494:Protein Kinases; C422202:spkA protein, Synechocystis; D013917:Thrombin