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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

Immunoblot showing tyrosine phosphorylation of wild-type Wzd, ΔWzd and Wze proteins expressed inL. lactissubsp.cremorisMG1363. Detection of tyrosine phosphorylation of Wze (27 kDa) along with wild type Wzd (33 kDa) or nine separate tyrosine mutated Wzd versions (33 kDa) expressed in L. lactis subsp. cremoris MG1363 with plasmids pDY33FEB (lane 1), pDY44FEB (lane 2), pDY77FEB (lane 3), pDY110FEB (lane 4), pDY114FEB (lane 5), pDY124FEB (lane 6), pDY134FEB (lane 7), pDY266FEB (lane 8), pDY141FEB (lane 9), pDWTEB (lane 10). Cell lysates of L. lactis subsp. cremoris MG1363 transformants were separated by 12% SDS-PAGE and tyrosine phosphorylated proteins were probed with mouse monoclonal anti-phosphotyrosine antibody.
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Fig1: Immunoblot showing tyrosine phosphorylation of wild-type Wzd, ΔWzd and Wze proteins expressed inL. lactissubsp.cremorisMG1363. Detection of tyrosine phosphorylation of Wze (27 kDa) along with wild type Wzd (33 kDa) or nine separate tyrosine mutated Wzd versions (33 kDa) expressed in L. lactis subsp. cremoris MG1363 with plasmids pDY33FEB (lane 1), pDY44FEB (lane 2), pDY77FEB (lane 3), pDY110FEB (lane 4), pDY114FEB (lane 5), pDY124FEB (lane 6), pDY134FEB (lane 7), pDY266FEB (lane 8), pDY141FEB (lane 9), pDWTEB (lane 10). Cell lysates of L. lactis subsp. cremoris MG1363 transformants were separated by 12% SDS-PAGE and tyrosine phosphorylated proteins were probed with mouse monoclonal anti-phosphotyrosine antibody.

Mentions: Nine mutated wzd genes changing a different tyrosine codon to phenylalanine were expressed individually in concert with wze and wzb from L. rhamnosus RW-9595 M in L. lactis subsp. cremoris MG1363 carrying one of the nine constructed plasmids (Table 1). As negative controls, there was no phosphorylated protein when extracts of L. lactis subsp. cremoris MG1363 with or without plasmid pMG36CT were probed with the anti-phosphotyrosine antibody (data not shown). All nine separate tyrosine mutated Wzd proteins (33 kDa) were phosphorylated (Figure 1). For six mutants (Figure 1, Lanes 1 to 6), band density was approximately equivalent to the wild-type Wzd (ratio of 1, as determined by ImageJ densitometry analysis). Two mutant proteins had slightly less band density than wild-type Wzd (Figure 1, Lane 10) with ratios of 0.9 (Wzd mutant Y134F; Lane 7) and 0.78 (Wzd mutant Y141F; Lane 9). The WzdY266F protein had the lowest ratio of 0.54 (Figure 1, lane 8) with respect to wild-type Wzd. Wze (27 kDa) was also phosphorylated when co-expressed with each of the nine mutated Wzd proteins in L. lactis subsp. cremoris MG1363 (Figure 1), albeit to a lower level when Wzd carried mutations Y33F, Y266F and Y241F (Figure 1, Lanes 1, 8 and 9).Figure 1


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)

Immunoblot showing tyrosine phosphorylation of wild-type Wzd, ΔWzd and Wze proteins expressed inL. lactissubsp.cremorisMG1363. Detection of tyrosine phosphorylation of Wze (27 kDa) along with wild type Wzd (33 kDa) or nine separate tyrosine mutated Wzd versions (33 kDa) expressed in L. lactis subsp. cremoris MG1363 with plasmids pDY33FEB (lane 1), pDY44FEB (lane 2), pDY77FEB (lane 3), pDY110FEB (lane 4), pDY114FEB (lane 5), pDY124FEB (lane 6), pDY134FEB (lane 7), pDY266FEB (lane 8), pDY141FEB (lane 9), pDWTEB (lane 10). Cell lysates of L. lactis subsp. cremoris MG1363 transformants were separated by 12% SDS-PAGE and tyrosine phosphorylated proteins were probed with mouse monoclonal anti-phosphotyrosine antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340800&req=5

Fig1: Immunoblot showing tyrosine phosphorylation of wild-type Wzd, ΔWzd and Wze proteins expressed inL. lactissubsp.cremorisMG1363. Detection of tyrosine phosphorylation of Wze (27 kDa) along with wild type Wzd (33 kDa) or nine separate tyrosine mutated Wzd versions (33 kDa) expressed in L. lactis subsp. cremoris MG1363 with plasmids pDY33FEB (lane 1), pDY44FEB (lane 2), pDY77FEB (lane 3), pDY110FEB (lane 4), pDY114FEB (lane 5), pDY124FEB (lane 6), pDY134FEB (lane 7), pDY266FEB (lane 8), pDY141FEB (lane 9), pDWTEB (lane 10). Cell lysates of L. lactis subsp. cremoris MG1363 transformants were separated by 12% SDS-PAGE and tyrosine phosphorylated proteins were probed with mouse monoclonal anti-phosphotyrosine antibody.
Mentions: Nine mutated wzd genes changing a different tyrosine codon to phenylalanine were expressed individually in concert with wze and wzb from L. rhamnosus RW-9595 M in L. lactis subsp. cremoris MG1363 carrying one of the nine constructed plasmids (Table 1). As negative controls, there was no phosphorylated protein when extracts of L. lactis subsp. cremoris MG1363 with or without plasmid pMG36CT were probed with the anti-phosphotyrosine antibody (data not shown). All nine separate tyrosine mutated Wzd proteins (33 kDa) were phosphorylated (Figure 1). For six mutants (Figure 1, Lanes 1 to 6), band density was approximately equivalent to the wild-type Wzd (ratio of 1, as determined by ImageJ densitometry analysis). Two mutant proteins had slightly less band density than wild-type Wzd (Figure 1, Lane 10) with ratios of 0.9 (Wzd mutant Y134F; Lane 7) and 0.78 (Wzd mutant Y141F; Lane 9). The WzdY266F protein had the lowest ratio of 0.54 (Figure 1, lane 8) with respect to wild-type Wzd. Wze (27 kDa) was also phosphorylated when co-expressed with each of the nine mutated Wzd proteins in L. lactis subsp. cremoris MG1363 (Figure 1), albeit to a lower level when Wzd carried mutations Y33F, Y266F and Y241F (Figure 1, Lanes 1, 8 and 9).Figure 1

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