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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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Related in: MedlinePlus

Proteome of soluble fractions of Synechocystis sp. PCC 6803. Soluble proteins (50 μg) from wild-type GS cells grown at 32°C (A) and wild-type GS cells treated for 30 min at 44°C (B) were separated by 2DE and stained with Colloidal Coomassie Briliant Blue G-250. Corresponding autoradiographs (C and D). Dried gels were exposed to X-ray films for 30 h at –70°C. Spot numbers of the identified proteins correspond to those presented in Table 1.
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DSR006F2: Proteome of soluble fractions of Synechocystis sp. PCC 6803. Soluble proteins (50 μg) from wild-type GS cells grown at 32°C (A) and wild-type GS cells treated for 30 min at 44°C (B) were separated by 2DE and stained with Colloidal Coomassie Briliant Blue G-250. Corresponding autoradiographs (C and D). Dried gels were exposed to X-ray films for 30 h at –70°C. Spot numbers of the identified proteins correspond to those presented in Table 1.

Mentions: Short-term heat stress had no significant influence on soluble protein composition as demonstrated by 2D gels stained with colloidal CBB G-250 (Fig. 2A and B). However, the extent of phosphorylation of the individual polypeptides changed upon heat treatment (Fig. 2C and D). The phosphorylation signals from polypeptides presented by spots 1 and 2 increased twice, whereas the phosphorylation signal from spots 3а/3b decreased 1.5 times. Phosphopeptide 4 did not show any temperature-dependent change in phosphorylation. The high-intensity signal from phosphopeptide 5 dropped significantly upon an increase in temperature from 32 to 44°C (Fig. 2C and D).Figure 2.


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)

Proteome of soluble fractions of Synechocystis sp. PCC 6803. Soluble proteins (50 μg) from wild-type GS cells grown at 32°C (A) and wild-type GS cells treated for 30 min at 44°C (B) were separated by 2DE and stained with Colloidal Coomassie Briliant Blue G-250. Corresponding autoradiographs (C and D). Dried gels were exposed to X-ray films for 30 h at –70°C. Spot numbers of the identified proteins correspond to those presented in Table 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DSR006F2: Proteome of soluble fractions of Synechocystis sp. PCC 6803. Soluble proteins (50 μg) from wild-type GS cells grown at 32°C (A) and wild-type GS cells treated for 30 min at 44°C (B) were separated by 2DE and stained with Colloidal Coomassie Briliant Blue G-250. Corresponding autoradiographs (C and D). Dried gels were exposed to X-ray films for 30 h at –70°C. Spot numbers of the identified proteins correspond to those presented in Table 1.
Mentions: Short-term heat stress had no significant influence on soluble protein composition as demonstrated by 2D gels stained with colloidal CBB G-250 (Fig. 2A and B). However, the extent of phosphorylation of the individual polypeptides changed upon heat treatment (Fig. 2C and D). The phosphorylation signals from polypeptides presented by spots 1 and 2 increased twice, whereas the phosphorylation signal from spots 3а/3b decreased 1.5 times. Phosphopeptide 4 did not show any temperature-dependent change in phosphorylation. The high-intensity signal from phosphopeptide 5 dropped significantly upon an increase in temperature from 32 to 44°C (Fig. 2C and D).Figure 2.

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