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The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Kim SY, Bender KW, Walker BJ, Zielinski RE, Spalding MH, Ort DR, Huber SC - Front Plant Sci (2016)

Bottom Line: Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco.In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit.Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements.

View Article: PubMed Central - PubMed

Affiliation: Global Change and Photosynthesis Research Unit, United States Department of Agriculture - Agricultural Research Service, UrbanaIL, USA; Plant Biology, University of Illinois at Champaign-Urbana, UrbanaIL, USA.

ABSTRACT
Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

No MeSH data available.


Related in: MedlinePlus

Plastid CK2α is the major protein kinase responsible for phosphorylation of RCA at the Thr-78 site. The cpck2 mutant has dramatically reduced phosphorylation of RCA at the Thr-78 site following (A) transfer of plants from the light to dark or (B) treatment of leaves with H2O2 for 1 h in the light. (C) Phosphorylation of recombinant RCAβ with recombinant cpCK2α in vitro confirms the involvement of CK2α. In each blot, the bands corresponding to the α- and β-isoforms, as appropriate, are indicated. The red asterisk in the anti-pT78 blot in (A) indicates an off-target signal that serves as a loading control. (D) Semi-quantitative RT-PCR analysis of the T-DNA insertion line, GABI400A04. Primers complementary to sequences flanking the insertion site in the cpCK2 mRNA were used. Primers within the ubiquitin (UBQ) coding sequence were used as a positive control.
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Figure 5: Plastid CK2α is the major protein kinase responsible for phosphorylation of RCA at the Thr-78 site. The cpck2 mutant has dramatically reduced phosphorylation of RCA at the Thr-78 site following (A) transfer of plants from the light to dark or (B) treatment of leaves with H2O2 for 1 h in the light. (C) Phosphorylation of recombinant RCAβ with recombinant cpCK2α in vitro confirms the involvement of CK2α. In each blot, the bands corresponding to the α- and β-isoforms, as appropriate, are indicated. The red asterisk in the anti-pT78 blot in (A) indicates an off-target signal that serves as a loading control. (D) Semi-quantitative RT-PCR analysis of the T-DNA insertion line, GABI400A04. Primers complementary to sequences flanking the insertion site in the cpCK2 mRNA were used. Primers within the ubiquitin (UBQ) coding sequence were used as a positive control.

Mentions: The sequence surrounding RCA-Thr78 is flanked by Asp residues suggesting that CK2 may be the requisite kinase. Chloroplasts are known to contain one member of the CK2 family known ascpCK2α, which is thought to be a major protein kinase in the plastid. To test the possible role of cpCK2α in RCA phosphorylation, we obtained a homozygous T-DNA mutant (GABI400A04) with the insert in the fifth exon of the gene that allowed us to test the function of this protein kinase in vivo. Indeed, disruption of cpCK2α substantially reduced phosphorylation of RCA at the Thr-78 site relative to wild type plants when leaves were darkened (Figure 5A), or when phosphorylation of RCA was induced in response to H2O2 feeding in the light (Figure 5B). These results suggest that cpCK2α is the major kinase involved in phosphorylation of RCA at Thr-78 in vivo. To test the role of cpCK2α further, we produced recombinant cpCK2α in E. coli for in vitro phosphorylation assays using recombinant RCAβ as the substrate. As shown in Figure 5C, recombinant cpCK2α-His6 phosphorylated recombinant RCAβ at the Thr-78 site in vitro, consistent with its suggested role in vivo. Phosphorylation of RCA was dependent upon cpCK2α and as is characteristic of the entire CK2 family (Niefind et al., 1999), cpCK2α required Mg2+ but could utilize ATP or GTP as co-substrate in the phosphorylation reaction. These results are consistent with the general notion that cpCK2α is the cognate kinase involved in RCA phosphorylation in vivo. Inactivation of the cpCK2 gene in the T-DNA insertional line used in these experiments was tested using semi-quantitative reverse transcription (RT)-PCR. As shown in Figure 5D, the mRNA for cpCK2 was present in wild type Columbia plants but was not present in the insertional mutant, confirming the knockout phenotype.


The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Kim SY, Bender KW, Walker BJ, Zielinski RE, Spalding MH, Ort DR, Huber SC - Front Plant Sci (2016)

Plastid CK2α is the major protein kinase responsible for phosphorylation of RCA at the Thr-78 site. The cpck2 mutant has dramatically reduced phosphorylation of RCA at the Thr-78 site following (A) transfer of plants from the light to dark or (B) treatment of leaves with H2O2 for 1 h in the light. (C) Phosphorylation of recombinant RCAβ with recombinant cpCK2α in vitro confirms the involvement of CK2α. In each blot, the bands corresponding to the α- and β-isoforms, as appropriate, are indicated. The red asterisk in the anti-pT78 blot in (A) indicates an off-target signal that serves as a loading control. (D) Semi-quantitative RT-PCR analysis of the T-DNA insertion line, GABI400A04. Primers complementary to sequences flanking the insertion site in the cpCK2 mRNA were used. Primers within the ubiquitin (UBQ) coding sequence were used as a positive control.
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Figure 5: Plastid CK2α is the major protein kinase responsible for phosphorylation of RCA at the Thr-78 site. The cpck2 mutant has dramatically reduced phosphorylation of RCA at the Thr-78 site following (A) transfer of plants from the light to dark or (B) treatment of leaves with H2O2 for 1 h in the light. (C) Phosphorylation of recombinant RCAβ with recombinant cpCK2α in vitro confirms the involvement of CK2α. In each blot, the bands corresponding to the α- and β-isoforms, as appropriate, are indicated. The red asterisk in the anti-pT78 blot in (A) indicates an off-target signal that serves as a loading control. (D) Semi-quantitative RT-PCR analysis of the T-DNA insertion line, GABI400A04. Primers complementary to sequences flanking the insertion site in the cpCK2 mRNA were used. Primers within the ubiquitin (UBQ) coding sequence were used as a positive control.
Mentions: The sequence surrounding RCA-Thr78 is flanked by Asp residues suggesting that CK2 may be the requisite kinase. Chloroplasts are known to contain one member of the CK2 family known ascpCK2α, which is thought to be a major protein kinase in the plastid. To test the possible role of cpCK2α in RCA phosphorylation, we obtained a homozygous T-DNA mutant (GABI400A04) with the insert in the fifth exon of the gene that allowed us to test the function of this protein kinase in vivo. Indeed, disruption of cpCK2α substantially reduced phosphorylation of RCA at the Thr-78 site relative to wild type plants when leaves were darkened (Figure 5A), or when phosphorylation of RCA was induced in response to H2O2 feeding in the light (Figure 5B). These results suggest that cpCK2α is the major kinase involved in phosphorylation of RCA at Thr-78 in vivo. To test the role of cpCK2α further, we produced recombinant cpCK2α in E. coli for in vitro phosphorylation assays using recombinant RCAβ as the substrate. As shown in Figure 5C, recombinant cpCK2α-His6 phosphorylated recombinant RCAβ at the Thr-78 site in vitro, consistent with its suggested role in vivo. Phosphorylation of RCA was dependent upon cpCK2α and as is characteristic of the entire CK2 family (Niefind et al., 1999), cpCK2α required Mg2+ but could utilize ATP or GTP as co-substrate in the phosphorylation reaction. These results are consistent with the general notion that cpCK2α is the cognate kinase involved in RCA phosphorylation in vivo. Inactivation of the cpCK2 gene in the T-DNA insertional line used in these experiments was tested using semi-quantitative reverse transcription (RT)-PCR. As shown in Figure 5D, the mRNA for cpCK2 was present in wild type Columbia plants but was not present in the insertional mutant, confirming the knockout phenotype.

Bottom Line: Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco.In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit.Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements.

View Article: PubMed Central - PubMed

Affiliation: Global Change and Photosynthesis Research Unit, United States Department of Agriculture - Agricultural Research Service, UrbanaIL, USA; Plant Biology, University of Illinois at Champaign-Urbana, UrbanaIL, USA.

ABSTRACT
Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

No MeSH data available.


Related in: MedlinePlus