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A tyrosine phosphorylation switch controls the interaction between the transmembrane modulator protein Wzd and the tyrosine kinase Wze of Lactobacillus rhamnosus.

Kang HJ, Gilbert C, Badeaux F, Atlan D, LaPointe G - BMC Microbiol. (2015)

Bottom Line: Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form.This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd.The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

View Article: PubMed Central - PubMed

Affiliation: STELA Dairy Research Centre, INAF, Université Laval, Québec, G1V 0A6, QC, Canada. hjkang@ibs.re.kr.

ABSTRACT

Background: One proposed mechanism for assembly of secreted heteropolysaccharides by many Gram positive bacteria relies on the coordinated action of a polymerization complex through reversible phosphorylation events. The role of the tyrosine protein kinase transmembrane modulator is, however, not well understood.

Results: The protein sequences deduced from the wzb, wzd and wze genes from Lactobacillus rhamnosus ATCC 9595 and RW-9595 M contain motifs also found in corresponding proteins CpsB, CpsC and CpsD from Streptococcus pneumoniae D39 (serotype 2). Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form. When tyrosine 266 was mutated to phenylalanine, WzdY266F showed slightly less phosphorylated protein than those produced by using eight other tyrosine mutated Wzd genes, when expressed along with Wze and Wzb in Lactococcus lactis subsp. cremoris MG1363. In order to demonstrate the importance of ATP for the interactions among these proteins, native and fusion Wzb, Wzd and Wze proteins were expressed and purified from Escherichia coli cultures. The modulator protein, Wzd, binds with the phosphotyrosine kinase Wze, irrespective of its phosphorylation status. However, Wze attained a higher phosphorylation level after interacting with phosphorylated Wzd in the presence of 10 mM ATP. This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd.

Conclusion: The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

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Related in: MedlinePlus

In vitrointeraction assays between His6-Wzd (34 kDa) and GST-Wze (53 kDa) proteins. His6-Wzd bound to Ni2+ charged resin was incubated with the cell lysate of GST-Wze in the presence of 10 mM ATP (lanes 1, 4, 7 and 10) or absence of ATP (lanes 2, 5, 8 and 11). Lanes 3, 6, 9 and 12 contain native His6-Wzd alone on the Ni2+ resin. Interaction of GST-Wze with His6-Wzd expressed by E. coli BL21(DE3): (A) Coomassie stained 12% SDS-PAGE gel and (B) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody. Interaction of GST-Wze with resin-bound His6-Wzd expressed by E. coli C41(DE3) and treated with YOP (Yersinia tyrosine phosphatase): (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.
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Fig5: In vitrointeraction assays between His6-Wzd (34 kDa) and GST-Wze (53 kDa) proteins. His6-Wzd bound to Ni2+ charged resin was incubated with the cell lysate of GST-Wze in the presence of 10 mM ATP (lanes 1, 4, 7 and 10) or absence of ATP (lanes 2, 5, 8 and 11). Lanes 3, 6, 9 and 12 contain native His6-Wzd alone on the Ni2+ resin. Interaction of GST-Wze with His6-Wzd expressed by E. coli BL21(DE3): (A) Coomassie stained 12% SDS-PAGE gel and (B) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody. Interaction of GST-Wze with resin-bound His6-Wzd expressed by E. coli C41(DE3) and treated with YOP (Yersinia tyrosine phosphatase): (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.

Mentions: GST-Wze alone also did not bind to the Ni2+ resin in the absence of His6-Wzd (data not shown). A cell lysate of E. coli C41(DE3) pGSTWze was passed through a column containing purified His6-Wzd expressed by either E. coli BL21(DE3) (His6-WzdBL21) or C41(DE3) (His6-WzdC41) bound to the Ni2+ resin (Figure 5). GST-Wze was retained by His6-WzdBL21, both in the presence and absence of 10 mM ATP during incubation and washing (Figure 5A). In the presence of ATP, there was no discernible change of tyrosine phosphorylation of His6-WzdBL21 after the interaction (Figure 5B). GST-Wze was also retained by dephosphorylated His6-WzdC41, in the presence and absence of 10 mM ATP while YOP was retained as well (Figure 5C). YOP treatment did not affect protein migration and was not found in the wash fraction. In the presence or absence of ATP, His6-WzdC41 on the resin with GST-Wze was not phosphorylated (Figure 5D). Bound GST-Wze showed slightly more phosphorylated protein in the presence of ATP, but the difference does not appear significant and there was no discernible change in phosphorylation either before or after the interaction with His6-Wzd. The identical result was obtained using His6-WzdC41 without YOP treatment and GST-Wze.Figure 5


A tyrosine phosphorylation switch controls the interaction between the transmembrane modulator protein Wzd and the tyrosine kinase Wze of Lactobacillus rhamnosus.

Kang HJ, Gilbert C, Badeaux F, Atlan D, LaPointe G - BMC Microbiol. (2015)

In vitrointeraction assays between His6-Wzd (34 kDa) and GST-Wze (53 kDa) proteins. His6-Wzd bound to Ni2+ charged resin was incubated with the cell lysate of GST-Wze in the presence of 10 mM ATP (lanes 1, 4, 7 and 10) or absence of ATP (lanes 2, 5, 8 and 11). Lanes 3, 6, 9 and 12 contain native His6-Wzd alone on the Ni2+ resin. Interaction of GST-Wze with His6-Wzd expressed by E. coli BL21(DE3): (A) Coomassie stained 12% SDS-PAGE gel and (B) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody. Interaction of GST-Wze with resin-bound His6-Wzd expressed by E. coli C41(DE3) and treated with YOP (Yersinia tyrosine phosphatase): (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: In vitrointeraction assays between His6-Wzd (34 kDa) and GST-Wze (53 kDa) proteins. His6-Wzd bound to Ni2+ charged resin was incubated with the cell lysate of GST-Wze in the presence of 10 mM ATP (lanes 1, 4, 7 and 10) or absence of ATP (lanes 2, 5, 8 and 11). Lanes 3, 6, 9 and 12 contain native His6-Wzd alone on the Ni2+ resin. Interaction of GST-Wze with His6-Wzd expressed by E. coli BL21(DE3): (A) Coomassie stained 12% SDS-PAGE gel and (B) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody. Interaction of GST-Wze with resin-bound His6-Wzd expressed by E. coli C41(DE3) and treated with YOP (Yersinia tyrosine phosphatase): (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.
Mentions: GST-Wze alone also did not bind to the Ni2+ resin in the absence of His6-Wzd (data not shown). A cell lysate of E. coli C41(DE3) pGSTWze was passed through a column containing purified His6-Wzd expressed by either E. coli BL21(DE3) (His6-WzdBL21) or C41(DE3) (His6-WzdC41) bound to the Ni2+ resin (Figure 5). GST-Wze was retained by His6-WzdBL21, both in the presence and absence of 10 mM ATP during incubation and washing (Figure 5A). In the presence of ATP, there was no discernible change of tyrosine phosphorylation of His6-WzdBL21 after the interaction (Figure 5B). GST-Wze was also retained by dephosphorylated His6-WzdC41, in the presence and absence of 10 mM ATP while YOP was retained as well (Figure 5C). YOP treatment did not affect protein migration and was not found in the wash fraction. In the presence or absence of ATP, His6-WzdC41 on the resin with GST-Wze was not phosphorylated (Figure 5D). Bound GST-Wze showed slightly more phosphorylated protein in the presence of ATP, but the difference does not appear significant and there was no discernible change in phosphorylation either before or after the interaction with His6-Wzd. The identical result was obtained using His6-WzdC41 without YOP treatment and GST-Wze.Figure 5

Bottom Line: Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form.This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd.The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

View Article: PubMed Central - PubMed

Affiliation: STELA Dairy Research Centre, INAF, Université Laval, Québec, G1V 0A6, QC, Canada. hjkang@ibs.re.kr.

ABSTRACT

Background: One proposed mechanism for assembly of secreted heteropolysaccharides by many Gram positive bacteria relies on the coordinated action of a polymerization complex through reversible phosphorylation events. The role of the tyrosine protein kinase transmembrane modulator is, however, not well understood.

Results: The protein sequences deduced from the wzb, wzd and wze genes from Lactobacillus rhamnosus ATCC 9595 and RW-9595 M contain motifs also found in corresponding proteins CpsB, CpsC and CpsD from Streptococcus pneumoniae D39 (serotype 2). Use of an anti-phosphotyrosine antibody demonstrated that both Wzd and Wze can be found in tyrosine phosphorylated form. When tyrosine 266 was mutated to phenylalanine, WzdY266F showed slightly less phosphorylated protein than those produced by using eight other tyrosine mutated Wzd genes, when expressed along with Wze and Wzb in Lactococcus lactis subsp. cremoris MG1363. In order to demonstrate the importance of ATP for the interactions among these proteins, native and fusion Wzb, Wzd and Wze proteins were expressed and purified from Escherichia coli cultures. The modulator protein, Wzd, binds with the phosphotyrosine kinase Wze, irrespective of its phosphorylation status. However, Wze attained a higher phosphorylation level after interacting with phosphorylated Wzd in the presence of 10 mM ATP. This highly phosphorylated Wze did not remain in close association with phosphorylated Wzd.

Conclusion: The Wze tyrosine kinase protein of Lactobacillus rhamnosus thus carries out tyrosine phosphorylation of Wzd in addition to auto- and trans- phosphorylation of the kinase itself.

Show MeSH
Related in: MedlinePlus