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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

In vitro phosphorylation of XJTGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJTGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJTGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B. (F) Co-localization of the GFP:XJTGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP:XJTGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)
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Figure 3: In vitro phosphorylation of XJTGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJTGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJTGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B. (F) Co-localization of the GFP:XJTGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP:XJTGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)

Mentions: Based on the GTP results and phosphorylation predictions, we suspected that a CK2-like kinase might be responsible for phosphorylation of the XJTGB1 protein. Therefore, the CK2α subunits from N. benthamiana (NbCK2α) and barley (HvCK2α) were cloned, and the C-terminal His-tagged fusion proteins were purified from E. coli cells (Fig. 3A). Subsequent in vitro phosphorylation assays revealed that the purified NbCK2α and HvCK2α proteins efficiently phosphorylated the recombinant XJTGB1 protein in the presence of [γ-32P]ATP (Fig. 3B). CK2 is highly sensitive to heparin inhibition (Matsushita et al., 2003), and our results confirmed that the levels of phosphorylation were reduced proportionally with increasing heparin concentrations (Fig. 3C). To further test the kinase specificity, we used NbCK2α to compare XJTGB1 and TMV P30 MP (Ivanov et al., 2003) protein phosphorylation in the presence of [γ-32P]ATP and [γ-32P]GTP (Fig. 3D). In both cases, the NbCK2α phosphorylation assays resulted in the presence of highly intense bands that co-migrated with the XJTGB1 and P30 proteins, and the kinase exhibited similar activities in the presence of both ATP and GTP (Fig. 3D, lanes 3 and 4, and 6 and 7). Moreover, the absence of radioactive bands in reactions lacking the NbCK2α protein confirmed that the 58kDa XJTGB1 and the P30 proteins are not autophosphorylated and the lack of radioactivity in the reactions without substrate proteins also indicated that the NbCK2α protein is not self-phosphorylated (Fig. 3D, lanes 2 and 5). In addition, tests were carried out in the presence of Mn2+, Mg2+, and Ca2+ to assess the cation specificity of CK2 phosphorylation (Niefind et al., 1999). These results revealed that NbCK2α exhibited similar phosphorylation intensities for XJTGB1 and P30 in the presence of either Mn2+ or Mg2+ (Fig. 3E, lanes 2 and 3, and 7 and 8), and that 32P incorporation was negligible in reactions containing Ca2+ (Fig. 3E, lanes 4 and 9). All of the in vitro phosphorylation data with the TGB1 protein were consistent with several published biochemical properties of CK2 (Ivanov et al., 2003; Hung et al., 2014).


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)

In vitro phosphorylation of XJTGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJTGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJTGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B. (F) Co-localization of the GFP:XJTGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP:XJTGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)
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Related In: Results  -  Collection

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Figure 3: In vitro phosphorylation of XJTGB1 protein by recombinant CK2 kinase. (A) SDS-PAGE analysis of NbCK2α and HvCK2α purified from E. coli BL21 cells. (B) In vitro phosphorylation of XJTGB1 protein with the NbCK2α and HvCK2α recombinant proteins and negative controls lacking the kinases. (C) Effects of heparin on in vitro phosphorylation of XJTGB1 protein. Phosphorylation levels were reduced with increasing amount of heparin. (D) Ability of NbCK2α to use both ATP and GTP as phosphate donors. (E) Divalent metal ion specificity of NbCK2α and the TMV-MP (P30) proteins. The CBB-stained proteins at the bottom of panels (B)–(E) are as indicated as in Fig. 2B. (F) Co-localization of the GFP:XJTGB1 and DsRed:NbCK2α proteins in N. benthamiana leaf cells. Single localization of GFP:XJTGB1 and DsRed:NbCK2α proteins are indicated at the top of the panels. Bars, 50 μm. (This figure is available in colour at JXB online.)
Mentions: Based on the GTP results and phosphorylation predictions, we suspected that a CK2-like kinase might be responsible for phosphorylation of the XJTGB1 protein. Therefore, the CK2α subunits from N. benthamiana (NbCK2α) and barley (HvCK2α) were cloned, and the C-terminal His-tagged fusion proteins were purified from E. coli cells (Fig. 3A). Subsequent in vitro phosphorylation assays revealed that the purified NbCK2α and HvCK2α proteins efficiently phosphorylated the recombinant XJTGB1 protein in the presence of [γ-32P]ATP (Fig. 3B). CK2 is highly sensitive to heparin inhibition (Matsushita et al., 2003), and our results confirmed that the levels of phosphorylation were reduced proportionally with increasing heparin concentrations (Fig. 3C). To further test the kinase specificity, we used NbCK2α to compare XJTGB1 and TMV P30 MP (Ivanov et al., 2003) protein phosphorylation in the presence of [γ-32P]ATP and [γ-32P]GTP (Fig. 3D). In both cases, the NbCK2α phosphorylation assays resulted in the presence of highly intense bands that co-migrated with the XJTGB1 and P30 proteins, and the kinase exhibited similar activities in the presence of both ATP and GTP (Fig. 3D, lanes 3 and 4, and 6 and 7). Moreover, the absence of radioactive bands in reactions lacking the NbCK2α protein confirmed that the 58kDa XJTGB1 and the P30 proteins are not autophosphorylated and the lack of radioactivity in the reactions without substrate proteins also indicated that the NbCK2α protein is not self-phosphorylated (Fig. 3D, lanes 2 and 5). In addition, tests were carried out in the presence of Mn2+, Mg2+, and Ca2+ to assess the cation specificity of CK2 phosphorylation (Niefind et al., 1999). These results revealed that NbCK2α exhibited similar phosphorylation intensities for XJTGB1 and P30 in the presence of either Mn2+ or Mg2+ (Fig. 3E, lanes 2 and 3, and 7 and 8), and that 32P incorporation was negligible in reactions containing Ca2+ (Fig. 3E, lanes 4 and 9). All of the in vitro phosphorylation data with the TGB1 protein were consistent with several published biochemical properties of CK2 (Ivanov et al., 2003; Hung et al., 2014).

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