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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 of the recombinant GroES protein by soluble protein fractions obtained from Synechocystis and E. coli. (A) Soluble protein fraction was obtained from disrupted cells of Synechocystis GS, which had been grown at 32°C and transferred to 44°C for 30 min, by centrifugation at 100 000g for 1 h. Soluble protein fraction of E. coli BL21cells transformed with pET-GroES was obtained from disrupted cells by centrifugation at 16 000g for 20 min. Cells were grown at 37°C, and the protein extracts were obtained from non-induced cells, and from cells, in which the expression of GroES was induced by 50 mM IPTG for 3 h. About 1.8 μg of the purified recombinant GroES was added to each protein fraction (7.5 μg) in the presence of 1.5 μCi of [γ-32P]ATP. The reaction of phosphorylation was carried out for 15 min at 30°C. Upper panel represents the results of phosphorylation of externally provided GroES. Lower panel shows parts of gels stained with CBB.
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DSR006F4: Phosphorylation of the recombinant GroES protein by soluble protein fractions obtained from Synechocystis and E. coli. (A) Soluble protein fraction was obtained from disrupted cells of Synechocystis GS, which had been grown at 32°C and transferred to 44°C for 30 min, by centrifugation at 100 000g for 1 h. Soluble protein fraction of E. coli BL21cells transformed with pET-GroES was obtained from disrupted cells by centrifugation at 16 000g for 20 min. Cells were grown at 37°C, and the protein extracts were obtained from non-induced cells, and from cells, in which the expression of GroES was induced by 50 mM IPTG for 3 h. About 1.8 μg of the purified recombinant GroES was added to each protein fraction (7.5 μg) in the presence of 1.5 μCi of [γ-32P]ATP. The reaction of phosphorylation was carried out for 15 min at 30°C. Upper panel represents the results of phosphorylation of externally provided GroES. Lower panel shows parts of gels stained with CBB.

Mentions: To ensure that the recombinant GroES is not phosphorylated by E. coli cells, we compared the patterns of phosphorylation obtained with Synechocystis and E. coli-soluble protein extracts. Figure 4 demonstrates that E. coli cells did not phosphorylate expressed and/or externally provided recombinant GroES protein. Thus, we conclude that phosphorylation of GroES demonstrated in Fig. 3 entirely results from the activity of the cyanobacterial STPKs.Figure 4.


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 of the recombinant GroES protein by soluble protein fractions obtained from Synechocystis and E. coli. (A) Soluble protein fraction was obtained from disrupted cells of Synechocystis GS, which had been grown at 32°C and transferred to 44°C for 30 min, by centrifugation at 100 000g for 1 h. Soluble protein fraction of E. coli BL21cells transformed with pET-GroES was obtained from disrupted cells by centrifugation at 16 000g for 20 min. Cells were grown at 37°C, and the protein extracts were obtained from non-induced cells, and from cells, in which the expression of GroES was induced by 50 mM IPTG for 3 h. About 1.8 μg of the purified recombinant GroES was added to each protein fraction (7.5 μg) in the presence of 1.5 μCi of [γ-32P]ATP. The reaction of phosphorylation was carried out for 15 min at 30°C. Upper panel represents the results of phosphorylation of externally provided GroES. Lower panel shows parts of gels stained with CBB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DSR006F4: Phosphorylation of the recombinant GroES protein by soluble protein fractions obtained from Synechocystis and E. coli. (A) Soluble protein fraction was obtained from disrupted cells of Synechocystis GS, which had been grown at 32°C and transferred to 44°C for 30 min, by centrifugation at 100 000g for 1 h. Soluble protein fraction of E. coli BL21cells transformed with pET-GroES was obtained from disrupted cells by centrifugation at 16 000g for 20 min. Cells were grown at 37°C, and the protein extracts were obtained from non-induced cells, and from cells, in which the expression of GroES was induced by 50 mM IPTG for 3 h. About 1.8 μg of the purified recombinant GroES was added to each protein fraction (7.5 μg) in the presence of 1.5 μCi of [γ-32P]ATP. The reaction of phosphorylation was carried out for 15 min at 30°C. Upper panel represents the results of phosphorylation of externally provided GroES. Lower panel shows parts of gels stained with CBB.
Mentions: To ensure that the recombinant GroES is not phosphorylated by E. coli cells, we compared the patterns of phosphorylation obtained with Synechocystis and E. coli-soluble protein extracts. Figure 4 demonstrates that E. coli cells did not phosphorylate expressed and/or externally provided recombinant GroES protein. Thus, we conclude that phosphorylation of GroES demonstrated in Fig. 3 entirely results from the activity of the cyanobacterial STPKs.Figure 4.

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