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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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).

Figure 4.

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.

Figure 5.

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.

Figure 6.

Phosphorylation in vitro of soluble protein fraction isolated from Synechocystis GS ΔHrcA mutant grown at 32°C and treated at 44°C for 30 min. Autoradiographs of phosphoprotein patterns at 32°C (A) and 44°C (B) and the corresponding western blots (C and D) developed with anti-Hsp60 (GroEL) antibodies are presented. Dashed circles designate the positions of GroEL. Dried gels were exposed to X-ray films for 20 h at –70°C.

Figure 7.

Phosphorylation in vitro of soluble protein fraction isolated from Synechocystis GT ΔHik34 mutant grown at 32°C and treated at 44°C for 30 min. Autoradiographs of phosphoprotein patterns at 32°C (A) and 44°C (B) and the corresponding western blots (C and D) developed with anti-Hsp60 (GroEL) antibodies are presented. Dashed circles designate the positions of GroEL. Dried gels were exposed to X-ray films for 30 h at −70°C.