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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

Detection of tyrosine phosphorylated GST-Wze released in the wash fractions. GST-Wze in wash fractions in the presence of 10 mM ATP (1–3 and 7–9) and absence of ATP (4–6 and 10–12) from interaction of GST-Wze with resin-bound 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. GST-Wze in wash fractions in the presence of 10 mM ATP (13–15 and 19–21) and absence of ATP (16–18 and 22–24) from interaction of GST-Wze with His6-Wzd expressed by E. coli C41: (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.
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Fig6: Detection of tyrosine phosphorylated GST-Wze released in the wash fractions. GST-Wze in wash fractions in the presence of 10 mM ATP (1–3 and 7–9) and absence of ATP (4–6 and 10–12) from interaction of GST-Wze with resin-bound 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. GST-Wze in wash fractions in the presence of 10 mM ATP (13–15 and 19–21) and absence of ATP (16–18 and 22–24) from interaction of GST-Wze with His6-Wzd expressed by E. coli C41: (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.

Mentions: In contrast, the phosphorylation state of GST-Wze in the wash fractions was very different from that bound to His6-Wzd on the Ni2+ resin. For the same amount of protein (Figure 6A, lanes 1, 2 and 3), GST-Wze was found in the wash fraction with a high phosphorylation level (Figure 6B, lanes 7, 8 and 9), after interacting with His6-WzdBL21 in the presence of ATP. In the absence of ATP, the phosphorylated GST-Wze signal in the wash fraction was much lower (Figure 6B, lanes 10 to 12) for the same amount of protein (Figure 6A, lanes 4 to 6). There appears to be some phosphorylated GST-Wze retained by His6-Wzd, but the highly phosphorylated GST-Wze did not bind strongly to Wzd. After interacting with His6-WzdC41, GST-Wze was washed out of the resin, and the low level phosphorylation does not appear to significantly differ in the presence or absence of ATP (Figure 6C and D).Figure 6


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)

Detection of tyrosine phosphorylated GST-Wze released in the wash fractions. GST-Wze in wash fractions in the presence of 10 mM ATP (1–3 and 7–9) and absence of ATP (4–6 and 10–12) from interaction of GST-Wze with resin-bound 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. GST-Wze in wash fractions in the presence of 10 mM ATP (13–15 and 19–21) and absence of ATP (16–18 and 22–24) from interaction of GST-Wze with His6-Wzd expressed by E. coli C41: (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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getmorefigures.php?uid=PMC4340800&req=5

Fig6: Detection of tyrosine phosphorylated GST-Wze released in the wash fractions. GST-Wze in wash fractions in the presence of 10 mM ATP (1–3 and 7–9) and absence of ATP (4–6 and 10–12) from interaction of GST-Wze with resin-bound 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. GST-Wze in wash fractions in the presence of 10 mM ATP (13–15 and 19–21) and absence of ATP (16–18 and 22–24) from interaction of GST-Wze with His6-Wzd expressed by E. coli C41: (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.
Mentions: In contrast, the phosphorylation state of GST-Wze in the wash fractions was very different from that bound to His6-Wzd on the Ni2+ resin. For the same amount of protein (Figure 6A, lanes 1, 2 and 3), GST-Wze was found in the wash fraction with a high phosphorylation level (Figure 6B, lanes 7, 8 and 9), after interacting with His6-WzdBL21 in the presence of ATP. In the absence of ATP, the phosphorylated GST-Wze signal in the wash fraction was much lower (Figure 6B, lanes 10 to 12) for the same amount of protein (Figure 6A, lanes 4 to 6). There appears to be some phosphorylated GST-Wze retained by His6-Wzd, but the highly phosphorylated GST-Wze did not bind strongly to Wzd. After interacting with His6-WzdC41, GST-Wze was washed out of the resin, and the low level phosphorylation does not appear to significantly differ in the presence or absence of ATP (Figure 6C and D).Figure 6

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