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Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

Hu Y, Li Z, Yuan C, Jin X, Yan L, Zhao X, Zhang Y, Jackson AO, Wang X, Han C, Yu J, Li D - J. Exp. Bot. (2015)

Bottom Line: Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections.The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability.Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

View Article: PubMed Central - PubMed

Affiliation: State Key laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.

No MeSH data available.


Related in: MedlinePlus

Thr-401 is the major XJTGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJTGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJTGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJTGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ-32P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B. (C) Phosphorylation comparisons of selected XJTGB1 protein mutants with wt XJTGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)
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Figure 4: Thr-401 is the major XJTGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJTGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJTGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJTGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ-32P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B. (C) Phosphorylation comparisons of selected XJTGB1 protein mutants with wt XJTGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)

Mentions: To identify the phosphorylation sites of CK2, the purified XJTGB1 protein was phosphorylated by NbCK2α in vitro with unlabelled ATP, and the gel-purified phosphorylated and unphosphorylated XJTGB1 proteins were separated by PAGE and digested with trypsin. The trypsin digestion products were analysed by Q-Exactive LC-MS/MS. The analysis showed that 87.9% of the TGB1 protein amino acid sequence was covered, and revealed that the phosphorylated and unphosphorylated proteins differed in a unique monophosphorylated peptide (399GETDETEKNIAFTVDTVR416) with a 2103.9362 m/z peak corresponding to a neutral precursor ion lacking phosphoric acid (97.9769Da). Based on the observed masses of the phosphorylated and unphosphorylated y16 fragment ions (Fig. 4A), we conclude that the phosphorylated XJTGB1 residue is located at Thr-401.


Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

Hu Y, Li Z, Yuan C, Jin X, Yan L, Zhao X, Zhang Y, Jackson AO, Wang X, Han C, Yu J, Li D - J. Exp. Bot. (2015)

Thr-401 is the major XJTGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJTGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJTGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJTGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ-32P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B. (C) Phosphorylation comparisons of selected XJTGB1 protein mutants with wt XJTGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)
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Figure 4: Thr-401 is the major XJTGB1 protein site for CK2 phosphorylation. (A) LC-MS/MS analysis of XJTGB1 protein phosphorylation by NbCK2α. The absence of phosphoric acid (97.9769Da) on the y16 ion fragment demonstrates that Thr-401 is a phosphorylation site for CK2 kinase. (B) Identification of the phosphorylation sites in XJTGB1 protein mutants by in vitro phosphorylation with HvCK2α and NbCK2α. The radioactive intensities of the XJTGB1 protein and its phosphorylation mutants indicate the extent of radiolabelling with [γ-32P]ATP. CBB-stained proteins at the bottom of the panels (B) and (C) are as indicated in Fig. 2B. (C) Phosphorylation comparisons of selected XJTGB1 protein mutants with wt XJTGB1 protein to confirm that Thr-401 is the major phosphorylated residue. (This figure is available in colour at JXB online.)
Mentions: To identify the phosphorylation sites of CK2, the purified XJTGB1 protein was phosphorylated by NbCK2α in vitro with unlabelled ATP, and the gel-purified phosphorylated and unphosphorylated XJTGB1 proteins were separated by PAGE and digested with trypsin. The trypsin digestion products were analysed by Q-Exactive LC-MS/MS. The analysis showed that 87.9% of the TGB1 protein amino acid sequence was covered, and revealed that the phosphorylated and unphosphorylated proteins differed in a unique monophosphorylated peptide (399GETDETEKNIAFTVDTVR416) with a 2103.9362 m/z peak corresponding to a neutral precursor ion lacking phosphoric acid (97.9769Da). Based on the observed masses of the phosphorylated and unphosphorylated y16 fragment ions (Fig. 4A), we conclude that the phosphorylated XJTGB1 residue is located at Thr-401.

Bottom Line: Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections.The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability.Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

View Article: PubMed Central - PubMed

Affiliation: State Key laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.

No MeSH data available.


Related in: MedlinePlus