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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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Stability of phosphorylated GroES at different pHs. (A) Autoradiographs of the phosphorylated GroES. Purified GroES (2.5 μg) mixed with either protein extracts derived from control (32°C) wild-type cells (10 μg) (lanes 1, 3, and 5) or with heat-treated (44°C for 30 min) cells (10 μg) (lanes 2, 4, and 6) was phosphorylated in vitro with [γ-32P]ATP. The reaction was terminated with 3× concentrated SDS–PAGE sample buffer and immediately subjected to SDS–PAGE (15% PAG). After electrophoresis, the proteins were transferred onto PVDF membrane. The membranes were stained with Ponceau red and incubated at 45°C for 2 h in 50 mM KCl–HCl (pH 1.0), 0.1 M Tris–HCl (pH 7), or 1 M KOH (pH 14). The radioactivity remaining in the membrane was revealed after its exposure onto a X-ray film. (B). Immunoblots of proteins from the wild-type cells of Synechocystis probed with monoclonal antibodies against phosphorylated Ser and Thr (anit-P-Thr and anti-P-Ser). Left panel: Protein extracts (25 μg) isolated from control (lane 1) and heat-treated (lane 2) wild-type cells were probed with anti-P-Thr antibodies. Central panel: Protein extracts (25 μg) isolated from control (lanes 3 and 5) and heat-treated (lanes 4 and 6) wild-type cells were probed with anti-P-Thr antibodies after phosphorylation in vitro with exogenously added recombinant GroES (2.5 μg). Right panel: The same as the middle panel but probed with anti-P-Ser antibodies.
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DSR006F5: Stability of phosphorylated GroES at different pHs. (A) Autoradiographs of the phosphorylated GroES. Purified GroES (2.5 μg) mixed with either protein extracts derived from control (32°C) wild-type cells (10 μg) (lanes 1, 3, and 5) or with heat-treated (44°C for 30 min) cells (10 μg) (lanes 2, 4, and 6) was phosphorylated in vitro with [γ-32P]ATP. The reaction was terminated with 3× concentrated SDS–PAGE sample buffer and immediately subjected to SDS–PAGE (15% PAG). After electrophoresis, the proteins were transferred onto PVDF membrane. The membranes were stained with Ponceau red and incubated at 45°C for 2 h in 50 mM KCl–HCl (pH 1.0), 0.1 M Tris–HCl (pH 7), or 1 M KOH (pH 14). The radioactivity remaining in the membrane was revealed after its exposure onto a X-ray film. (B). Immunoblots of proteins from the wild-type cells of Synechocystis probed with monoclonal antibodies against phosphorylated Ser and Thr (anit-P-Thr and anti-P-Ser). Left panel: Protein extracts (25 μg) isolated from control (lane 1) and heat-treated (lane 2) wild-type cells were probed with anti-P-Thr antibodies. Central panel: Protein extracts (25 μg) isolated from control (lanes 3 and 5) and heat-treated (lanes 4 and 6) wild-type cells were probed with anti-P-Thr antibodies after phosphorylation in vitro with exogenously added recombinant GroES (2.5 μg). Right panel: The same as the middle panel but probed with anti-P-Ser antibodies.

Mentions: Proteins were isolated from control and heat-stressed wild-type cells, phosphorylated with [γ-32P]ATP, separated by electrophoresis, blotted onto PVDF, and stained with Ponceau red. The portions of the membrane containing the GroES were incubated in solutions with different pH: 1.0, 7.0, and 14.0. A majority of phosphate incorporated into GroES was released when the incubation was carried out at 45°C for 2 h at pH 14 (Fig. 5A). N-Phosphate amino acids (phoshohistidine, phospholysine, and phosphoarginine) are known to be stable at pH of 14.0 but not at pH of 1.0, while O-phosphate amino acids (phosphoserine and phosphothreonine) are known to be stable at pH of 1.0 but not at pH of 14.0.49 On the other hand, acyl-phosphate amino acids (phosphoaspartate and phosphoglutamate) are labile at both pH of 1.0 and 14.0, whereas the phosphotyrosine and phosphocysteine are stable at both pH. We observed a disappearance of the radioactive signal from the blots at pH of 14.0. Therefore, the stability profile for phosphorylated GroES is in agreement with Ser/Thr nature of protein phosphorylation (Fig. 5A).Figure 5.


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)

Stability of phosphorylated GroES at different pHs. (A) Autoradiographs of the phosphorylated GroES. Purified GroES (2.5 μg) mixed with either protein extracts derived from control (32°C) wild-type cells (10 μg) (lanes 1, 3, and 5) or with heat-treated (44°C for 30 min) cells (10 μg) (lanes 2, 4, and 6) was phosphorylated in vitro with [γ-32P]ATP. The reaction was terminated with 3× concentrated SDS–PAGE sample buffer and immediately subjected to SDS–PAGE (15% PAG). After electrophoresis, the proteins were transferred onto PVDF membrane. The membranes were stained with Ponceau red and incubated at 45°C for 2 h in 50 mM KCl–HCl (pH 1.0), 0.1 M Tris–HCl (pH 7), or 1 M KOH (pH 14). The radioactivity remaining in the membrane was revealed after its exposure onto a X-ray film. (B). Immunoblots of proteins from the wild-type cells of Synechocystis probed with monoclonal antibodies against phosphorylated Ser and Thr (anit-P-Thr and anti-P-Ser). Left panel: Protein extracts (25 μg) isolated from control (lane 1) and heat-treated (lane 2) wild-type cells were probed with anti-P-Thr antibodies. Central panel: Protein extracts (25 μg) isolated from control (lanes 3 and 5) and heat-treated (lanes 4 and 6) wild-type cells were probed with anti-P-Thr antibodies after phosphorylation in vitro with exogenously added recombinant GroES (2.5 μg). Right panel: The same as the middle panel but probed with anti-P-Ser antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DSR006F5: Stability of phosphorylated GroES at different pHs. (A) Autoradiographs of the phosphorylated GroES. Purified GroES (2.5 μg) mixed with either protein extracts derived from control (32°C) wild-type cells (10 μg) (lanes 1, 3, and 5) or with heat-treated (44°C for 30 min) cells (10 μg) (lanes 2, 4, and 6) was phosphorylated in vitro with [γ-32P]ATP. The reaction was terminated with 3× concentrated SDS–PAGE sample buffer and immediately subjected to SDS–PAGE (15% PAG). After electrophoresis, the proteins were transferred onto PVDF membrane. The membranes were stained with Ponceau red and incubated at 45°C for 2 h in 50 mM KCl–HCl (pH 1.0), 0.1 M Tris–HCl (pH 7), or 1 M KOH (pH 14). The radioactivity remaining in the membrane was revealed after its exposure onto a X-ray film. (B). Immunoblots of proteins from the wild-type cells of Synechocystis probed with monoclonal antibodies against phosphorylated Ser and Thr (anit-P-Thr and anti-P-Ser). Left panel: Protein extracts (25 μg) isolated from control (lane 1) and heat-treated (lane 2) wild-type cells were probed with anti-P-Thr antibodies. Central panel: Protein extracts (25 μg) isolated from control (lanes 3 and 5) and heat-treated (lanes 4 and 6) wild-type cells were probed with anti-P-Thr antibodies after phosphorylation in vitro with exogenously added recombinant GroES (2.5 μg). Right panel: The same as the middle panel but probed with anti-P-Ser antibodies.
Mentions: Proteins were isolated from control and heat-stressed wild-type cells, phosphorylated with [γ-32P]ATP, separated by electrophoresis, blotted onto PVDF, and stained with Ponceau red. The portions of the membrane containing the GroES were incubated in solutions with different pH: 1.0, 7.0, and 14.0. A majority of phosphate incorporated into GroES was released when the incubation was carried out at 45°C for 2 h at pH 14 (Fig. 5A). N-Phosphate amino acids (phoshohistidine, phospholysine, and phosphoarginine) are known to be stable at pH of 14.0 but not at pH of 1.0, while O-phosphate amino acids (phosphoserine and phosphothreonine) are known to be stable at pH of 1.0 but not at pH of 14.0.49 On the other hand, acyl-phosphate amino acids (phosphoaspartate and phosphoglutamate) are labile at both pH of 1.0 and 14.0, whereas the phosphotyrosine and phosphocysteine are stable at both pH. We observed a disappearance of the radioactive signal from the blots at pH of 14.0. Therefore, the stability profile for phosphorylated GroES is in agreement with Ser/Thr nature of protein phosphorylation (Fig. 5A).Figure 5.

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