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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

Model proposed for the tyrosine switch controlling EPS polymerization and attachment. 1. Non-phosphorylated Wzd and Wze interact by forming a complex leading to release of EPS (+) rather than attachment (−). 2. In the presence of ATP, Wze phosphorylates Wzd, allowing chain elongation by the polymerase (+). This phosphorylation of Wzd destabilizes the protein interaction with Wze, allowing Wze to either bind with other non-phosphorylated Wzd proteins, or undergo auto- or trans-transphosphorylation. 3. A transitory interaction between phosphor-rylated Wzd and Wze in the presence of ATP is necessary for the phosphorylation of Wze, possibly allowing the attachment of polysaccharide to the cell wall (+). Phosphatase activity of Wzb for dephosphorylating Wzd and Wze would return the cycle to the non-phosphorylated Wzd/Wze complex that allows release of polysaccharide polymers (+). Question marks indicate proposed steps.
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Fig7: Model proposed for the tyrosine switch controlling EPS polymerization and attachment. 1. Non-phosphorylated Wzd and Wze interact by forming a complex leading to release of EPS (+) rather than attachment (−). 2. In the presence of ATP, Wze phosphorylates Wzd, allowing chain elongation by the polymerase (+). This phosphorylation of Wzd destabilizes the protein interaction with Wze, allowing Wze to either bind with other non-phosphorylated Wzd proteins, or undergo auto- or trans-transphosphorylation. 3. A transitory interaction between phosphor-rylated Wzd and Wze in the presence of ATP is necessary for the phosphorylation of Wze, possibly allowing the attachment of polysaccharide to the cell wall (+). Phosphatase activity of Wzb for dephosphorylating Wzd and Wze would return the cycle to the non-phosphorylated Wzd/Wze complex that allows release of polysaccharide polymers (+). Question marks indicate proposed steps.

Mentions: Our study shows that the activity of Wzd is also modulated through tyrosine phosphorylation of more than one tyrosine residue, allowing the phosphorylation of Wze. This new information suggests that further modification of the model for the control of EPS elongation can be proposed (Figure 7). The non-phosphorylated complex may allow polysaccharides to be released instead of attached to the cell wall. When Wze phosphorylates Wzd, interaction of Wzd with the polymerase may promote polysaccharide elongation [14]. Destabilization of the protein interaction between Wzd and Wze liberates Wze to interact with other non-phosphorylated Wzd or Wze proteins. When Wzd is phosphorylated and ATP is present, a transitory interaction allows Wze to autophosphorylate or transphosphorylate other Wze proteins, possibly slowing polymerization and leading to the attachment of polysaccharide to the cell wall if a ligase is present.Figure 7


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)

Model proposed for the tyrosine switch controlling EPS polymerization and attachment. 1. Non-phosphorylated Wzd and Wze interact by forming a complex leading to release of EPS (+) rather than attachment (−). 2. In the presence of ATP, Wze phosphorylates Wzd, allowing chain elongation by the polymerase (+). This phosphorylation of Wzd destabilizes the protein interaction with Wze, allowing Wze to either bind with other non-phosphorylated Wzd proteins, or undergo auto- or trans-transphosphorylation. 3. A transitory interaction between phosphor-rylated Wzd and Wze in the presence of ATP is necessary for the phosphorylation of Wze, possibly allowing the attachment of polysaccharide to the cell wall (+). Phosphatase activity of Wzb for dephosphorylating Wzd and Wze would return the cycle to the non-phosphorylated Wzd/Wze complex that allows release of polysaccharide polymers (+). Question marks indicate proposed steps.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Model proposed for the tyrosine switch controlling EPS polymerization and attachment. 1. Non-phosphorylated Wzd and Wze interact by forming a complex leading to release of EPS (+) rather than attachment (−). 2. In the presence of ATP, Wze phosphorylates Wzd, allowing chain elongation by the polymerase (+). This phosphorylation of Wzd destabilizes the protein interaction with Wze, allowing Wze to either bind with other non-phosphorylated Wzd proteins, or undergo auto- or trans-transphosphorylation. 3. A transitory interaction between phosphor-rylated Wzd and Wze in the presence of ATP is necessary for the phosphorylation of Wze, possibly allowing the attachment of polysaccharide to the cell wall (+). Phosphatase activity of Wzb for dephosphorylating Wzd and Wze would return the cycle to the non-phosphorylated Wzd/Wze complex that allows release of polysaccharide polymers (+). Question marks indicate proposed steps.
Mentions: Our study shows that the activity of Wzd is also modulated through tyrosine phosphorylation of more than one tyrosine residue, allowing the phosphorylation of Wze. This new information suggests that further modification of the model for the control of EPS elongation can be proposed (Figure 7). The non-phosphorylated complex may allow polysaccharides to be released instead of attached to the cell wall. When Wze phosphorylates Wzd, interaction of Wzd with the polymerase may promote polysaccharide elongation [14]. Destabilization of the protein interaction between Wzd and Wze liberates Wze to interact with other non-phosphorylated Wzd or Wze proteins. When Wzd is phosphorylated and ATP is present, a transitory interaction allows Wze to autophosphorylate or transphosphorylate other Wze proteins, possibly slowing polymerization and leading to the attachment of polysaccharide to the cell wall if a ligase is present.Figure 7

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