Limits...
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.


Effect of sequence alterations in C4 domain on phosphorylation and protein interaction.(a) In vitro kinase assay with wild-type (WT) or L277Q versions of C4 peptide (Fig. 1a) fused to GST or GST alone as a substrate.32P and CBB panels show autoradiography and Coomassie Brilliant Blue (CBB) staining images of parts of the gel. Substrate (Sub) and loading control (LC, showing a band around the molecular mass of RubisCO large subunit) panels indicate that similar amounts of substrates and protein extracts from shoot apices of Day 7 plants, respectively, were used in the experiment. (b) Effect of sequence alterations in the C4 domain on the interaction with FT and 14-3-3s, examined by yeast two-hybrid assay. Wild-type (FD), L277Q, and LQTE (L277Q T282E) versions of FD were tested for an interaction with FT and two isoforms of 14-3-3, GRF3 (14-3-3 psi) and GRF4 (14-3-3 phi). AD and BD indicate the activation domain and DNA binding domain, respectively, of GAL4. –LWH and –LW indicate selective (SCD –Leu, –Trp, –His) and non-selective (SCD –Leu, –Trp) medium, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4321167&req=5

f3: Effect of sequence alterations in C4 domain on phosphorylation and protein interaction.(a) In vitro kinase assay with wild-type (WT) or L277Q versions of C4 peptide (Fig. 1a) fused to GST or GST alone as a substrate.32P and CBB panels show autoradiography and Coomassie Brilliant Blue (CBB) staining images of parts of the gel. Substrate (Sub) and loading control (LC, showing a band around the molecular mass of RubisCO large subunit) panels indicate that similar amounts of substrates and protein extracts from shoot apices of Day 7 plants, respectively, were used in the experiment. (b) Effect of sequence alterations in the C4 domain on the interaction with FT and 14-3-3s, examined by yeast two-hybrid assay. Wild-type (FD), L277Q, and LQTE (L277Q T282E) versions of FD were tested for an interaction with FT and two isoforms of 14-3-3, GRF3 (14-3-3 psi) and GRF4 (14-3-3 phi). AD and BD indicate the activation domain and DNA binding domain, respectively, of GAL4. –LWH and –LW indicate selective (SCD –Leu, –Trp, –His) and non-selective (SCD –Leu, –Trp) medium, respectively.

Mentions: To test whether CDPK phosphorylates FD T282, we analyzed substrate sequence specificity of protein kinases against FD C-terminal peptides. CDPK requires a hydrophobic amino acid residue at the −5 position for phosphorylation, especially leucine32. In other words, CDPK recognizes an L-X-R/K-X-X-S/T sequence and phosphorylates serine or threonine within this motif. The C-terminal sequence of L-Q-R-S-S-T in FD and its orthologous proteins conforms to this rule (Supplementary Fig. S3a). Thus, if CDPK is responsible for phosphorylation of FD T282, substitution of a leucine at the −5 position (L277) to glutamine (L277Q) should abolish phosphorylation. An in vitro kinase assay with a L277Q substitution in the C4 peptide (Fig. 1a) as a substrate showed that protein kinases in extracts of shoot apices failed to phosphorylate the C4 peptide with L277Q (Fig. 3a). Furthermore, L277Q mFD failed to interact with FT and 14-3-3s (GRF3 and GRF4) in yeast two-hybrid assays (Fig. 3b). As expected, phospho-mimic substitution (T282E) in L277Q mFD (termed LQTE) resulted in restoration of interaction with FT and 14-3-3 (Fig. 3b). These results indicate that L277 is crucial for phosphorylation of T282 and thereby the interaction with FT and 14-3-3s. To evaluate the activity in planta, we generated and analyzed transgenic plants expressing various mutant FD proteins under the control of the cauliflower mosaic virus (CaMV) 35S RNA (35S) promoter in an fd-1 background. In contrast to wild-type FD and phospho-mimic mutant FD (T282E mFD and LQTE mFD), which were able to rescue fd-1, non-phosphorylatable T282A mFD failed to rescue fd-1, and L277Q mFD showed reduced complementation ability (Supplementary Fig. S3b). Thus, the complementation tests indicate the importance of L277 for FD function in planta. Taken together, the biochemical characterization of substrate specificity (Fig. 3) and in planta complementation assays (Supplementary Fig. S3b) strongly suggest that CDPK phosphorylates FD T282 and is required for the FD-FT florigen protein complex formation.


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)

Effect of sequence alterations in C4 domain on phosphorylation and protein interaction.(a) In vitro kinase assay with wild-type (WT) or L277Q versions of C4 peptide (Fig. 1a) fused to GST or GST alone as a substrate.32P and CBB panels show autoradiography and Coomassie Brilliant Blue (CBB) staining images of parts of the gel. Substrate (Sub) and loading control (LC, showing a band around the molecular mass of RubisCO large subunit) panels indicate that similar amounts of substrates and protein extracts from shoot apices of Day 7 plants, respectively, were used in the experiment. (b) Effect of sequence alterations in the C4 domain on the interaction with FT and 14-3-3s, examined by yeast two-hybrid assay. Wild-type (FD), L277Q, and LQTE (L277Q T282E) versions of FD were tested for an interaction with FT and two isoforms of 14-3-3, GRF3 (14-3-3 psi) and GRF4 (14-3-3 phi). AD and BD indicate the activation domain and DNA binding domain, respectively, of GAL4. –LWH and –LW indicate selective (SCD –Leu, –Trp, –His) and non-selective (SCD –Leu, –Trp) medium, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321167&req=5

f3: Effect of sequence alterations in C4 domain on phosphorylation and protein interaction.(a) In vitro kinase assay with wild-type (WT) or L277Q versions of C4 peptide (Fig. 1a) fused to GST or GST alone as a substrate.32P and CBB panels show autoradiography and Coomassie Brilliant Blue (CBB) staining images of parts of the gel. Substrate (Sub) and loading control (LC, showing a band around the molecular mass of RubisCO large subunit) panels indicate that similar amounts of substrates and protein extracts from shoot apices of Day 7 plants, respectively, were used in the experiment. (b) Effect of sequence alterations in the C4 domain on the interaction with FT and 14-3-3s, examined by yeast two-hybrid assay. Wild-type (FD), L277Q, and LQTE (L277Q T282E) versions of FD were tested for an interaction with FT and two isoforms of 14-3-3, GRF3 (14-3-3 psi) and GRF4 (14-3-3 phi). AD and BD indicate the activation domain and DNA binding domain, respectively, of GAL4. –LWH and –LW indicate selective (SCD –Leu, –Trp, –His) and non-selective (SCD –Leu, –Trp) medium, respectively.
Mentions: To test whether CDPK phosphorylates FD T282, we analyzed substrate sequence specificity of protein kinases against FD C-terminal peptides. CDPK requires a hydrophobic amino acid residue at the −5 position for phosphorylation, especially leucine32. In other words, CDPK recognizes an L-X-R/K-X-X-S/T sequence and phosphorylates serine or threonine within this motif. The C-terminal sequence of L-Q-R-S-S-T in FD and its orthologous proteins conforms to this rule (Supplementary Fig. S3a). Thus, if CDPK is responsible for phosphorylation of FD T282, substitution of a leucine at the −5 position (L277) to glutamine (L277Q) should abolish phosphorylation. An in vitro kinase assay with a L277Q substitution in the C4 peptide (Fig. 1a) as a substrate showed that protein kinases in extracts of shoot apices failed to phosphorylate the C4 peptide with L277Q (Fig. 3a). Furthermore, L277Q mFD failed to interact with FT and 14-3-3s (GRF3 and GRF4) in yeast two-hybrid assays (Fig. 3b). As expected, phospho-mimic substitution (T282E) in L277Q mFD (termed LQTE) resulted in restoration of interaction with FT and 14-3-3 (Fig. 3b). These results indicate that L277 is crucial for phosphorylation of T282 and thereby the interaction with FT and 14-3-3s. To evaluate the activity in planta, we generated and analyzed transgenic plants expressing various mutant FD proteins under the control of the cauliflower mosaic virus (CaMV) 35S RNA (35S) promoter in an fd-1 background. In contrast to wild-type FD and phospho-mimic mutant FD (T282E mFD and LQTE mFD), which were able to rescue fd-1, non-phosphorylatable T282A mFD failed to rescue fd-1, and L277Q mFD showed reduced complementation ability (Supplementary Fig. S3b). Thus, the complementation tests indicate the importance of L277 for FD function in planta. Taken together, the biochemical characterization of substrate specificity (Fig. 3) and in planta complementation assays (Supplementary Fig. S3b) strongly suggest that CDPK phosphorylates FD T282 and is required for the FD-FT florigen protein complex formation.

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.