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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

Western immunoblot of the phosphorylation state of His6-Wzd and GST-Wze proteins expressed inE. coli. Tyrosine phosphorylation of His6-Wzd and GST-Wze was detected by using Western immunoblotting with a mouse monoclonal antibody. Coomassie stained 12% SDS-PAGE gel of His6-Wzd expressed in E. coli BL21(DE3) (A) and C41(DE3) (B): incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (C) Detection of tyrosine phosphorylated His6-Wzd expressed in E. coli BL21(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (D) Absence of detection of tyrosine phosphorylated His6-Wzd expressed in E. coli C41(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (E) Detection of tyrosine phosphorylated GST-Wze expressed in E. coli C41(DE3) incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2).
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Fig3: Western immunoblot of the phosphorylation state of His6-Wzd and GST-Wze proteins expressed inE. coli. Tyrosine phosphorylation of His6-Wzd and GST-Wze was detected by using Western immunoblotting with a mouse monoclonal antibody. Coomassie stained 12% SDS-PAGE gel of His6-Wzd expressed in E. coli BL21(DE3) (A) and C41(DE3) (B): incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (C) Detection of tyrosine phosphorylated His6-Wzd expressed in E. coli BL21(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (D) Absence of detection of tyrosine phosphorylated His6-Wzd expressed in E. coli C41(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (E) Detection of tyrosine phosphorylated GST-Wze expressed in E. coli C41(DE3) incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2).

Mentions: The phosphorylation state of the expressed proteins was verified prior to testing protein interactions. To detect any autophosphorylation, proteins were incubated with or without ATP. As controls, cell lysates of the host strains BL21(DE3) and C41(DE3) carrying the vector plasmids did not show any tyrosine phosphorylated proteins, with or without added ATP (data not shown). In the absence of added ATP, the His6-Wzd purified from E. coli BL21(DE3) was recognized by the anti-phosphotyrosine antibody, which shows tyrosine phosphorylation (Figure 3C). On the other hand, when purified after expression by strain C41(DE3), the similar amount of His6-Wzd was not phosphorylated after incubation either with or without ATP (Figure 3D), even though the protein was detected at by Western blot using the anti-His-tag antibody (data not shown). Under these conditions, the amount of phosphorylated His6-Wzd protein may be below the detection level of our assay.Figure 3


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)

Western immunoblot of the phosphorylation state of His6-Wzd and GST-Wze proteins expressed inE. coli. Tyrosine phosphorylation of His6-Wzd and GST-Wze was detected by using Western immunoblotting with a mouse monoclonal antibody. Coomassie stained 12% SDS-PAGE gel of His6-Wzd expressed in E. coli BL21(DE3) (A) and C41(DE3) (B): incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (C) Detection of tyrosine phosphorylated His6-Wzd expressed in E. coli BL21(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (D) Absence of detection of tyrosine phosphorylated His6-Wzd expressed in E. coli C41(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (E) Detection of tyrosine phosphorylated GST-Wze expressed in E. coli C41(DE3) incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340800&req=5

Fig3: Western immunoblot of the phosphorylation state of His6-Wzd and GST-Wze proteins expressed inE. coli. Tyrosine phosphorylation of His6-Wzd and GST-Wze was detected by using Western immunoblotting with a mouse monoclonal antibody. Coomassie stained 12% SDS-PAGE gel of His6-Wzd expressed in E. coli BL21(DE3) (A) and C41(DE3) (B): incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (C) Detection of tyrosine phosphorylated His6-Wzd expressed in E. coli BL21(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (D) Absence of detection of tyrosine phosphorylated His6-Wzd expressed in E. coli C41(DE3) and incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2). (E) Detection of tyrosine phosphorylated GST-Wze expressed in E. coli C41(DE3) incubated with 10 mM ATP (Lane 1) and without ATP (Lane 2).
Mentions: The phosphorylation state of the expressed proteins was verified prior to testing protein interactions. To detect any autophosphorylation, proteins were incubated with or without ATP. As controls, cell lysates of the host strains BL21(DE3) and C41(DE3) carrying the vector plasmids did not show any tyrosine phosphorylated proteins, with or without added ATP (data not shown). In the absence of added ATP, the His6-Wzd purified from E. coli BL21(DE3) was recognized by the anti-phosphotyrosine antibody, which shows tyrosine phosphorylation (Figure 3C). On the other hand, when purified after expression by strain C41(DE3), the similar amount of His6-Wzd was not phosphorylated after incubation either with or without ATP (Figure 3D), even though the protein was detected at by Western blot using the anti-His-tag antibody (data not shown). Under these conditions, the amount of phosphorylated His6-Wzd protein may be below the detection level of our assay.Figure 3

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