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Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation.

Kawamoto N, Sasabe M, Endo M, Machida Y, Araki T - Sci Rep (2015)

Bottom Line: The kinase activity was calcium-dependent.Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions.The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

ABSTRACT
Appropriate timing of flowering is critical for reproductive success and necessarily involves complex genetic regulatory networks. A mobile floral signal, called florigen, is a key molecule in this process, and flowering locus T (FT) protein is its major component in Arabidopsis. FT is produced in leaves, but promotes the floral transition in the shoot apex, where it forms a complex with a basic region/leucine-zipper (bZIP) transcription factor, FD. Formation of the florigen complex depends on the supposed phosphorylation of FD; hitherto, however, the responsible protein kinase(s) have not been identified. In this study, we prepared protein extracts from shoot apices of plants around the floral transition, and detected a protein kinase activity that phosphorylates a threonine residue at position 282 of FD (FD T282), which is a crucial residue for the complex formation with FT via 14-3-3. The kinase activity was calcium-dependent. Subsequent biochemical, cellular, and genetic analyses showed that three calcium-dependent protein kinases (CDPKs) efficiently phosphorylate FD T282. Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions. The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.

No MeSH data available.


Related in: MedlinePlus

Biochemical characterization of candidate CDPKs.(a) Domain structure of CDPK and fragments used in this study. VN: variable N-terminal domain, AI: auto-inhibition domain, EF: EF hand motif. CPK-CAs were constructed based on published information38 and primers used for cloning are shown in Table S2. (b) In vitro phosphorylation of FD T282 by CPK-CAs. Wild-type (WT), T282A, and L277Q versions of C4 peptide (Fig. 1a) or GST were used as substrates. Lower panels (CBB staining) confirm similar amounts of substrates in the experiments. Uncropped images of autoradiograms are shown in Supplementary Fig. S5a. (c) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FD or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. One-tenth volumes of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6a (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6a (lower panels). (d) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FDP or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. Ten percent, by volume, of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6b (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6b (lower panels). (e) Calcium-dependency of kinase activity of CPK6 and CPK33. In vitro phosphorylation reactions used full-length purified CPK6 and CPK33. WT C4 peptide was used as a substrate. Phosphorylated and non-phosphorylated C4 peptides were separated by Phos-tag SDS-PAGE. White and black arrowheads indicate phosphorylated and non-phosphorylated C4 peptide, respectively.
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f4: Biochemical characterization of candidate CDPKs.(a) Domain structure of CDPK and fragments used in this study. VN: variable N-terminal domain, AI: auto-inhibition domain, EF: EF hand motif. CPK-CAs were constructed based on published information38 and primers used for cloning are shown in Table S2. (b) In vitro phosphorylation of FD T282 by CPK-CAs. Wild-type (WT), T282A, and L277Q versions of C4 peptide (Fig. 1a) or GST were used as substrates. Lower panels (CBB staining) confirm similar amounts of substrates in the experiments. Uncropped images of autoradiograms are shown in Supplementary Fig. S5a. (c) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FD or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. One-tenth volumes of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6a (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6a (lower panels). (d) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FDP or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. Ten percent, by volume, of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6b (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6b (lower panels). (e) Calcium-dependency of kinase activity of CPK6 and CPK33. In vitro phosphorylation reactions used full-length purified CPK6 and CPK33. WT C4 peptide was used as a substrate. Phosphorylated and non-phosphorylated C4 peptides were separated by Phos-tag SDS-PAGE. White and black arrowheads indicate phosphorylated and non-phosphorylated C4 peptide, respectively.

Mentions: In vitro kinase assays were performed to evaluate the enzymatic activity of candidate CDPKs on C4 peptide and truncated FD (tFD; amino acid residues 196–285) (Fig. 1a) as substrates (Supplementary Fig. S5). Constitutively active forms of CDPKs (CPK-CAs) lacking both the C-terminal auto-inhibition region and EF-hand motifs38 (Fig. 4a) were used for the assay. All the CPK-CAs except for CPK31 were expressed as soluble recombinant proteins and had kinase activity against a general substrate, myelin basic protein (MBP) (Supplementary Fig. S5a). Among the tested CDPKs, CPK4, CPK6, and CPK33 efficiently phosphorylated T282 in C4 peptide (Fig. 4b, Supplementary Fig. S5a). These three CDPKs and 5 other CDPKs that phosphorylated T282 in C4 peptide were further analyzed for their kinase activity against the tFD fragment containing the bZIP region for dimerization. CPK4, CPK6, and CPK33 efficiently phosphorylated T282 in tFD, as well. By contrast, the activity of CPK3, 5, 11, 27, and 32 was decreased or lost compared to the activity against C4 peptide (Supplementary Fig. S5b). These results suggest that CPK4, CPK6, and CPK33 are good candidates as FD kinases.


Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation.

Kawamoto N, Sasabe M, Endo M, Machida Y, Araki T - Sci Rep (2015)

Biochemical characterization of candidate CDPKs.(a) Domain structure of CDPK and fragments used in this study. VN: variable N-terminal domain, AI: auto-inhibition domain, EF: EF hand motif. CPK-CAs were constructed based on published information38 and primers used for cloning are shown in Table S2. (b) In vitro phosphorylation of FD T282 by CPK-CAs. Wild-type (WT), T282A, and L277Q versions of C4 peptide (Fig. 1a) or GST were used as substrates. Lower panels (CBB staining) confirm similar amounts of substrates in the experiments. Uncropped images of autoradiograms are shown in Supplementary Fig. S5a. (c) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FD or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. One-tenth volumes of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6a (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6a (lower panels). (d) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FDP or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. Ten percent, by volume, of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6b (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6b (lower panels). (e) Calcium-dependency of kinase activity of CPK6 and CPK33. In vitro phosphorylation reactions used full-length purified CPK6 and CPK33. WT C4 peptide was used as a substrate. Phosphorylated and non-phosphorylated C4 peptides were separated by Phos-tag SDS-PAGE. White and black arrowheads indicate phosphorylated and non-phosphorylated C4 peptide, respectively.
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Related In: Results  -  Collection

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Show All Figures
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f4: Biochemical characterization of candidate CDPKs.(a) Domain structure of CDPK and fragments used in this study. VN: variable N-terminal domain, AI: auto-inhibition domain, EF: EF hand motif. CPK-CAs were constructed based on published information38 and primers used for cloning are shown in Table S2. (b) In vitro phosphorylation of FD T282 by CPK-CAs. Wild-type (WT), T282A, and L277Q versions of C4 peptide (Fig. 1a) or GST were used as substrates. Lower panels (CBB staining) confirm similar amounts of substrates in the experiments. Uncropped images of autoradiograms are shown in Supplementary Fig. S5a. (c) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FD or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. One-tenth volumes of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6a (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6a (lower panels). (d) In vitro pull-down assay. Trx-His-CPKs were pulled-down with either GST-FDP or GST in the presence (+CaCl2) or absence (+EGTA) of Ca2+. Immunoblot with anti-His tag antibody. Ten percent, by volume, of the reactions were loaded in “10% input” lanes. Uncropped images are shown in Supplementary Fig. S6b (upper panels). Results of immunoblotting with anti-GST antibody are also shown in Supplementary Fig. S6b (lower panels). (e) Calcium-dependency of kinase activity of CPK6 and CPK33. In vitro phosphorylation reactions used full-length purified CPK6 and CPK33. WT C4 peptide was used as a substrate. Phosphorylated and non-phosphorylated C4 peptides were separated by Phos-tag SDS-PAGE. White and black arrowheads indicate phosphorylated and non-phosphorylated C4 peptide, respectively.
Mentions: In vitro kinase assays were performed to evaluate the enzymatic activity of candidate CDPKs on C4 peptide and truncated FD (tFD; amino acid residues 196–285) (Fig. 1a) as substrates (Supplementary Fig. S5). Constitutively active forms of CDPKs (CPK-CAs) lacking both the C-terminal auto-inhibition region and EF-hand motifs38 (Fig. 4a) were used for the assay. All the CPK-CAs except for CPK31 were expressed as soluble recombinant proteins and had kinase activity against a general substrate, myelin basic protein (MBP) (Supplementary Fig. S5a). Among the tested CDPKs, CPK4, CPK6, and CPK33 efficiently phosphorylated T282 in C4 peptide (Fig. 4b, Supplementary Fig. S5a). These three CDPKs and 5 other CDPKs that phosphorylated T282 in C4 peptide were further analyzed for their kinase activity against the tFD fragment containing the bZIP region for dimerization. CPK4, CPK6, and CPK33 efficiently phosphorylated T282 in tFD, as well. By contrast, the activity of CPK3, 5, 11, 27, and 32 was decreased or lost compared to the activity against C4 peptide (Supplementary Fig. S5b). These results suggest that CPK4, CPK6, and CPK33 are good candidates as FD kinases.

Bottom Line: The kinase activity was calcium-dependent.Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions.The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

ABSTRACT
Appropriate timing of flowering is critical for reproductive success and necessarily involves complex genetic regulatory networks. A mobile floral signal, called florigen, is a key molecule in this process, and flowering locus T (FT) protein is its major component in Arabidopsis. FT is produced in leaves, but promotes the floral transition in the shoot apex, where it forms a complex with a basic region/leucine-zipper (bZIP) transcription factor, FD. Formation of the florigen complex depends on the supposed phosphorylation of FD; hitherto, however, the responsible protein kinase(s) have not been identified. In this study, we prepared protein extracts from shoot apices of plants around the floral transition, and detected a protein kinase activity that phosphorylates a threonine residue at position 282 of FD (FD T282), which is a crucial residue for the complex formation with FT via 14-3-3. The kinase activity was calcium-dependent. Subsequent biochemical, cellular, and genetic analyses showed that three calcium-dependent protein kinases (CDPKs) efficiently phosphorylate FD T282. Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions. The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.

No MeSH data available.


Related in: MedlinePlus