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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

SDS-PAGE of IPTG-induced cell extracts and purified Wzd, Wzb and GST-Wze. (A) 34 kDa His6-Wzd fusion protein purification by affinity chromatography from E. coli C41(DE3) (pQEWzd) IPTG-induced culture lysate. Lane 2: The flow through contains proteins not retained by the Ni2+ column. Lanes 3 and 4: fractions from first and last column washes, respectively; Lane 5: fraction from the elution step. (B) Wzb and GST-Wze purified from IPTG-induced culture lysates of E. coli NM522 (pQEWzb) and E. coli BL21(DE3) (pGSTWze). Lanes 2 and 4: lysed culture supernatant; Lanes 3 and 5: purified Wzb (29 kDa) and GST-Wze (53 kDa) proteins. Lane 1: BenchMark Prestained Protein Ladder (Invitrogen).
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Fig2: SDS-PAGE of IPTG-induced cell extracts and purified Wzd, Wzb and GST-Wze. (A) 34 kDa His6-Wzd fusion protein purification by affinity chromatography from E. coli C41(DE3) (pQEWzd) IPTG-induced culture lysate. Lane 2: The flow through contains proteins not retained by the Ni2+ column. Lanes 3 and 4: fractions from first and last column washes, respectively; Lane 5: fraction from the elution step. (B) Wzb and GST-Wze purified from IPTG-induced culture lysates of E. coli NM522 (pQEWzb) and E. coli BL21(DE3) (pGSTWze). Lanes 2 and 4: lysed culture supernatant; Lanes 3 and 5: purified Wzb (29 kDa) and GST-Wze (53 kDa) proteins. Lane 1: BenchMark Prestained Protein Ladder (Invitrogen).

Mentions: Overexpression of the 34 kDa His6-tagged Wzd protein from the wzd gene of L. rhamnosus ATCC 9595 was achieved in two E. coli strains, namely BL21(DE3) or C41(DE3) carrying pQEWzd (Figure 2A). Untagged Wzb and Wze proteins of the predicted molecular mass were obtained in E. coli strain NM522 carrying either pQEWzb (Figure 1B) or pQEWze. GST-tagged Wze protein was obtained using both E. coli strains C41(DE3) or BL21(DE3) carrying pGSTWze (Figure 2B).Figure 2


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)

SDS-PAGE of IPTG-induced cell extracts and purified Wzd, Wzb and GST-Wze. (A) 34 kDa His6-Wzd fusion protein purification by affinity chromatography from E. coli C41(DE3) (pQEWzd) IPTG-induced culture lysate. Lane 2: The flow through contains proteins not retained by the Ni2+ column. Lanes 3 and 4: fractions from first and last column washes, respectively; Lane 5: fraction from the elution step. (B) Wzb and GST-Wze purified from IPTG-induced culture lysates of E. coli NM522 (pQEWzb) and E. coli BL21(DE3) (pGSTWze). Lanes 2 and 4: lysed culture supernatant; Lanes 3 and 5: purified Wzb (29 kDa) and GST-Wze (53 kDa) proteins. Lane 1: BenchMark Prestained Protein Ladder (Invitrogen).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: SDS-PAGE of IPTG-induced cell extracts and purified Wzd, Wzb and GST-Wze. (A) 34 kDa His6-Wzd fusion protein purification by affinity chromatography from E. coli C41(DE3) (pQEWzd) IPTG-induced culture lysate. Lane 2: The flow through contains proteins not retained by the Ni2+ column. Lanes 3 and 4: fractions from first and last column washes, respectively; Lane 5: fraction from the elution step. (B) Wzb and GST-Wze purified from IPTG-induced culture lysates of E. coli NM522 (pQEWzb) and E. coli BL21(DE3) (pGSTWze). Lanes 2 and 4: lysed culture supernatant; Lanes 3 and 5: purified Wzb (29 kDa) and GST-Wze (53 kDa) proteins. Lane 1: BenchMark Prestained Protein Ladder (Invitrogen).
Mentions: Overexpression of the 34 kDa His6-tagged Wzd protein from the wzd gene of L. rhamnosus ATCC 9595 was achieved in two E. coli strains, namely BL21(DE3) or C41(DE3) carrying pQEWzd (Figure 2A). Untagged Wzb and Wze proteins of the predicted molecular mass were obtained in E. coli strain NM522 carrying either pQEWzb (Figure 1B) or pQEWze. GST-tagged Wze protein was obtained using both E. coli strains C41(DE3) or BL21(DE3) carrying pGSTWze (Figure 2B).Figure 2

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