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Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the Cyanobacterium synechocystis.

Zorina A, Stepanchenko N, Novikova GV, Sinetova M, Panichkin VB, Moshkov IE, Zinchenko VV, Shestakov SV, Suzuki I, Murata N, Los DA - DNA Res. (2011)

Bottom Line: The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities.Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES.This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Physiology, Botanicheskaya Street 35, 127276 Moscow, Russia.

ABSTRACT
Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

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A schematic representation of the mutation of individual spk genes in Synechocystis by insertion of antibiotic-resistance gene cassettes. Open arrows represent the open reading frames (ORFs). Shaded arrows represent the target spk genes. Small black triangles indicate sites for binding of primers (see Supplementary Table S2) and directions of elongation of primers. The position and orientation of insertion of each cassette and the size and orientation of each respective gene are indicated in Supplementary Table S1.
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DSR006F1: A schematic representation of the mutation of individual spk genes in Synechocystis by insertion of antibiotic-resistance gene cassettes. Open arrows represent the open reading frames (ORFs). Shaded arrows represent the target spk genes. Small black triangles indicate sites for binding of primers (see Supplementary Table S2) and directions of elongation of primers. The position and orientation of insertion of each cassette and the size and orientation of each respective gene are indicated in Supplementary Table S1.

Mentions: Partial sequences of individual spk genes have been amplified by PCR with the primers listed in Supplementary Table S1. The amplified DNA fragments were ligated into pGEM®-T or pGEM®-T Easy (Promega Inc., Madison, WI, USA), or into pT7Blue-T, or pUC18.29 Antibiotic-resistance gene cassettes,30–33 a transposon EZ::TN™ <KAN-2> (Epicentre, Madison, WI, USA), or pGPS2.1 (New England BioLabs, Beverly, MA, USA) have been inserted into the cloned DNA fragments. Each of the resultant plasmids was used for transformation of Synechocystis cells. The transformed cells were selected on a agar-solidified BG11 medium that had been supplemented with an appropriate antibiotic, as described by Grigorieva and Shestakov.26 Each cyanobacterial cell contains several copies of the cyanobacterial chromosome.34,35 In order to mutate every copy of the chromosome, we transferred the transformed cells to several plates of the agar-solidified BG11 medium with increasing concentrations of the antibiotic. We confirmed the complete replacement of native copies of the chromosome by mutated copies by amplification by PCR (Supplementary Fig. S1) with the pairs of primers listed in Supplementary Table S2. Positions of insertions are shown in Fig. 1.Figure 1.


Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the Cyanobacterium synechocystis.

Zorina A, Stepanchenko N, Novikova GV, Sinetova M, Panichkin VB, Moshkov IE, Zinchenko VV, Shestakov SV, Suzuki I, Murata N, Los DA - DNA Res. (2011)

A schematic representation of the mutation of individual spk genes in Synechocystis by insertion of antibiotic-resistance gene cassettes. Open arrows represent the open reading frames (ORFs). Shaded arrows represent the target spk genes. Small black triangles indicate sites for binding of primers (see Supplementary Table S2) and directions of elongation of primers. The position and orientation of insertion of each cassette and the size and orientation of each respective gene are indicated in Supplementary Table S1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111230&req=5

DSR006F1: A schematic representation of the mutation of individual spk genes in Synechocystis by insertion of antibiotic-resistance gene cassettes. Open arrows represent the open reading frames (ORFs). Shaded arrows represent the target spk genes. Small black triangles indicate sites for binding of primers (see Supplementary Table S2) and directions of elongation of primers. The position and orientation of insertion of each cassette and the size and orientation of each respective gene are indicated in Supplementary Table S1.
Mentions: Partial sequences of individual spk genes have been amplified by PCR with the primers listed in Supplementary Table S1. The amplified DNA fragments were ligated into pGEM®-T or pGEM®-T Easy (Promega Inc., Madison, WI, USA), or into pT7Blue-T, or pUC18.29 Antibiotic-resistance gene cassettes,30–33 a transposon EZ::TN™ <KAN-2> (Epicentre, Madison, WI, USA), or pGPS2.1 (New England BioLabs, Beverly, MA, USA) have been inserted into the cloned DNA fragments. Each of the resultant plasmids was used for transformation of Synechocystis cells. The transformed cells were selected on a agar-solidified BG11 medium that had been supplemented with an appropriate antibiotic, as described by Grigorieva and Shestakov.26 Each cyanobacterial cell contains several copies of the cyanobacterial chromosome.34,35 In order to mutate every copy of the chromosome, we transferred the transformed cells to several plates of the agar-solidified BG11 medium with increasing concentrations of the antibiotic. We confirmed the complete replacement of native copies of the chromosome by mutated copies by amplification by PCR (Supplementary Fig. S1) with the pairs of primers listed in Supplementary Table S2. Positions of insertions are shown in Fig. 1.Figure 1.

Bottom Line: The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities.Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES.This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Physiology, Botanicheskaya Street 35, 127276 Moscow, Russia.

ABSTRACT
Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

Show MeSH
Related in: MedlinePlus