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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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Phosphorylation in vitro of recombinant GroES by soluble protein fractions isolated from Synechocystis sp. PCC6803 (GS), spk mutants, and some of the complemented mutants. (A) GroES was phosphorylated in vitro with protein extracts obtained from the wild-type cells of Synechocystis (GS) and spk mutants grown at 32°C, and incubated for 30 min at 44°C. Autoradiographs and Coomassie R-250-stained gel are presented. (B) Similar reactions have been performed with wild-type cells of Synechocystis (GS), and with the mutant strains defective in SpkF, SpkC, and SpkK, which have been complemented with pVZ-spkC, pVZ-spkF, and pVZ-spkK (designated as +spkC, +spkF, and +spkK, respectively).
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DSR006F3: Phosphorylation in vitro of recombinant GroES by soluble protein fractions isolated from Synechocystis sp. PCC6803 (GS), spk mutants, and some of the complemented mutants. (A) GroES was phosphorylated in vitro with protein extracts obtained from the wild-type cells of Synechocystis (GS) and spk mutants grown at 32°C, and incubated for 30 min at 44°C. Autoradiographs and Coomassie R-250-stained gel are presented. (B) Similar reactions have been performed with wild-type cells of Synechocystis (GS), and with the mutant strains defective in SpkF, SpkC, and SpkK, which have been complemented with pVZ-spkC, pVZ-spkF, and pVZ-spkK (designated as +spkC, +spkF, and +spkK, respectively).

Mentions: Wild-type and 11 mutant (ΔspkB-L) cells were treated at 44°C for 30 min, and soluble protein fractions were isolated from each strain. Twelve protein fractions isolated from those cells were equalized by protein content and used for phosphorylation of equal amounts of the purified recombinant GroES in the presence of [γ-32P]ATP. The products of reaction were resolved with SDS–PAGE, and phosphorylated GroES was visualized by autoradiography (Fig. 3A). Since equal amounts of isolated proteins were incubated with equal amounts of the recombinant GroES in the presence of equal amounts of [γ-32P]ATP, we can reliably validate the data on GroES phosphorylation presented in Fig. 3A.Figure 3.


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)

Phosphorylation in vitro of recombinant GroES by soluble protein fractions isolated from Synechocystis sp. PCC6803 (GS), spk mutants, and some of the complemented mutants. (A) GroES was phosphorylated in vitro with protein extracts obtained from the wild-type cells of Synechocystis (GS) and spk mutants grown at 32°C, and incubated for 30 min at 44°C. Autoradiographs and Coomassie R-250-stained gel are presented. (B) Similar reactions have been performed with wild-type cells of Synechocystis (GS), and with the mutant strains defective in SpkF, SpkC, and SpkK, which have been complemented with pVZ-spkC, pVZ-spkF, and pVZ-spkK (designated as +spkC, +spkF, and +spkK, respectively).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DSR006F3: Phosphorylation in vitro of recombinant GroES by soluble protein fractions isolated from Synechocystis sp. PCC6803 (GS), spk mutants, and some of the complemented mutants. (A) GroES was phosphorylated in vitro with protein extracts obtained from the wild-type cells of Synechocystis (GS) and spk mutants grown at 32°C, and incubated for 30 min at 44°C. Autoradiographs and Coomassie R-250-stained gel are presented. (B) Similar reactions have been performed with wild-type cells of Synechocystis (GS), and with the mutant strains defective in SpkF, SpkC, and SpkK, which have been complemented with pVZ-spkC, pVZ-spkF, and pVZ-spkK (designated as +spkC, +spkF, and +spkK, respectively).
Mentions: Wild-type and 11 mutant (ΔspkB-L) cells were treated at 44°C for 30 min, and soluble protein fractions were isolated from each strain. Twelve protein fractions isolated from those cells were equalized by protein content and used for phosphorylation of equal amounts of the purified recombinant GroES in the presence of [γ-32P]ATP. The products of reaction were resolved with SDS–PAGE, and phosphorylated GroES was visualized by autoradiography (Fig. 3A). Since equal amounts of isolated proteins were incubated with equal amounts of the recombinant GroES in the presence of equal amounts of [γ-32P]ATP, we can reliably validate the data on GroES phosphorylation presented in Fig. 3A.Figure 3.

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