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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

In vivo oxidation of the RCA α-isoform in the dark occurs rapidly and involves an intramolecular disulfide bond. Leaves were harvested at the indicated times and subjected by non-reducing SDS-PAGE followed by immunoblotting with anti-RCA antibodies. The bands corresponding to reduced- and oxidized-RCAα and the single RCAβ isoform are indicated; the black asterisk identifies the RbcL protein, which shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
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Figure 2: In vivo oxidation of the RCA α-isoform in the dark occurs rapidly and involves an intramolecular disulfide bond. Leaves were harvested at the indicated times and subjected by non-reducing SDS-PAGE followed by immunoblotting with anti-RCA antibodies. The bands corresponding to reduced- and oxidized-RCAα and the single RCAβ isoform are indicated; the black asterisk identifies the RbcL protein, which shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.

Mentions: In Arabidopsis, RCA is well known to be regulated by reversible disulfide bond formation between Cys-451 and Cys-470 located in the C-terminus that is unique to the α-isoform (Zhang and Portis, 1999). We reasoned that because the disulfide was intra- rather than inter-molecular, the redox modification may be observed as faster migration of the oxidized α-isoform on non-reducing SDS-PAGE. This has been noted in the past with other redox-regulated enzymes (Pollitt and Zalkin, 1983) and is attributed to the more compact structure of the oxidized polypeptide compared to the reduced polypeptide. As shown in Figure 2, two bands corresponding to RCAα could be resolved from leaves harvested in the light, whereas only a single band with faster migration was observed in leaves harvested in the dark. The migration of the β-isoform was unaffected by these treatments consistent with the notion that it does not contain redox-sensitive sulfhydryl groups (Zhang and Portis, 1999). It is important to note that inter-molecular disulfides of the α-isoform were not observed (see Supplementary Figure S2). However, blots prepared from non-reducing gels invariably showed greater non-specific reaction of the ∼50-kDa Rubisco large subunit (RbcL) protein (identified with the black asterisk in Figure 2 and Supplementary Figure S2).


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)

In vivo oxidation of the RCA α-isoform in the dark occurs rapidly and involves an intramolecular disulfide bond. Leaves were harvested at the indicated times and subjected by non-reducing SDS-PAGE followed by immunoblotting with anti-RCA antibodies. The bands corresponding to reduced- and oxidized-RCAα and the single RCAβ isoform are indicated; the black asterisk identifies the RbcL protein, which shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814456&req=5

Figure 2: In vivo oxidation of the RCA α-isoform in the dark occurs rapidly and involves an intramolecular disulfide bond. Leaves were harvested at the indicated times and subjected by non-reducing SDS-PAGE followed by immunoblotting with anti-RCA antibodies. The bands corresponding to reduced- and oxidized-RCAα and the single RCAβ isoform are indicated; the black asterisk identifies the RbcL protein, which shows increased antibody reaction when electrophoresis is performed under non-reducing conditions.
Mentions: In Arabidopsis, RCA is well known to be regulated by reversible disulfide bond formation between Cys-451 and Cys-470 located in the C-terminus that is unique to the α-isoform (Zhang and Portis, 1999). We reasoned that because the disulfide was intra- rather than inter-molecular, the redox modification may be observed as faster migration of the oxidized α-isoform on non-reducing SDS-PAGE. This has been noted in the past with other redox-regulated enzymes (Pollitt and Zalkin, 1983) and is attributed to the more compact structure of the oxidized polypeptide compared to the reduced polypeptide. As shown in Figure 2, two bands corresponding to RCAα could be resolved from leaves harvested in the light, whereas only a single band with faster migration was observed in leaves harvested in the dark. The migration of the β-isoform was unaffected by these treatments consistent with the notion that it does not contain redox-sensitive sulfhydryl groups (Zhang and Portis, 1999). It is important to note that inter-molecular disulfides of the α-isoform were not observed (see Supplementary Figure S2). However, blots prepared from non-reducing gels invariably showed greater non-specific reaction of the ∼50-kDa Rubisco large subunit (RbcL) protein (identified with the black asterisk in Figure 2 and Supplementary Figure S2).

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