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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

In vitrointeraction assay between resin-bound His6-WzdC41(34 kDa) and native Wze (27 kDa) proteins. Coomassie-stained gel A and B: (A) Lane 1 contains the BenchMark Prestained Protein Ladder (Invitrogen). Lane 2: The two proteins were incubated together in a 1:1 ratio with the Ni2+ charged resin for 4 h at 16°C in 2.5 ml buffer B1 containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 50 mM imidazole, 1% Triton, 5 mM MgCl2. Lanes 3 to 6: 5 ml wash fractions with buffer B1 in panels A and B (in panel B, 200 μM ATP was added to the wash in lanes 4 to 6). Lane 7: protein elution using 1 ml buffer containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 1 M imidazole, 1% Triton, 5 mM MgCl2, 2% sarcosyl. (C) Western blot of gel in panel B, detection was carried out using anti-phosphotyrosine antibody.
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Fig4: In vitrointeraction assay between resin-bound His6-WzdC41(34 kDa) and native Wze (27 kDa) proteins. Coomassie-stained gel A and B: (A) Lane 1 contains the BenchMark Prestained Protein Ladder (Invitrogen). Lane 2: The two proteins were incubated together in a 1:1 ratio with the Ni2+ charged resin for 4 h at 16°C in 2.5 ml buffer B1 containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 50 mM imidazole, 1% Triton, 5 mM MgCl2. Lanes 3 to 6: 5 ml wash fractions with buffer B1 in panels A and B (in panel B, 200 μM ATP was added to the wash in lanes 4 to 6). Lane 7: protein elution using 1 ml buffer containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 1 M imidazole, 1% Triton, 5 mM MgCl2, 2% sarcosyl. (C) Western blot of gel in panel B, detection was carried out using anti-phosphotyrosine antibody.

Mentions: Untagged Wzb and Wze were not retained by the nickel-charged affinity resin itself (data not shown), so they could be tested for affinity to resin-bound His6-Wzd. Wzb was not retained by His6-Wzd after washing the resin (data not shown), suggesting that no stable interaction was present between these two proteins. After incubating resin-bound His6-WzdC41 and Wze together, the resin was divided into two fractions for washing either with or without ATP. Wze was retained by His6-Wzd using the same washing conditions as with Wzb (Figure 4A). Without added ATP, the wash fraction did not contain any tyrosine-phosphorylated protein. However, when 200 μM ATP was added to the washing solution, Wze protein was released from resin-bound His6-Wzd in each of three successive washing steps (Figure 4B, lanes 4, 5 and 6). After eluting the remaining bound proteins from the same column, only a small quantity of non-phosphorylated Wze was associated with His6-Wzd (Figure 4B, Lane 7). Moreover, using the anti-phosphotyrosine antibody, the Wzd protein was revealed to be tyrosine-phosphorylated after washing with 200 μM ATP (Figure 4C).Figure 4


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)

In vitrointeraction assay between resin-bound His6-WzdC41(34 kDa) and native Wze (27 kDa) proteins. Coomassie-stained gel A and B: (A) Lane 1 contains the BenchMark Prestained Protein Ladder (Invitrogen). Lane 2: The two proteins were incubated together in a 1:1 ratio with the Ni2+ charged resin for 4 h at 16°C in 2.5 ml buffer B1 containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 50 mM imidazole, 1% Triton, 5 mM MgCl2. Lanes 3 to 6: 5 ml wash fractions with buffer B1 in panels A and B (in panel B, 200 μM ATP was added to the wash in lanes 4 to 6). Lane 7: protein elution using 1 ml buffer containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 1 M imidazole, 1% Triton, 5 mM MgCl2, 2% sarcosyl. (C) Western blot of gel in panel B, detection was carried out using anti-phosphotyrosine antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: In vitrointeraction assay between resin-bound His6-WzdC41(34 kDa) and native Wze (27 kDa) proteins. Coomassie-stained gel A and B: (A) Lane 1 contains the BenchMark Prestained Protein Ladder (Invitrogen). Lane 2: The two proteins were incubated together in a 1:1 ratio with the Ni2+ charged resin for 4 h at 16°C in 2.5 ml buffer B1 containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 50 mM imidazole, 1% Triton, 5 mM MgCl2. Lanes 3 to 6: 5 ml wash fractions with buffer B1 in panels A and B (in panel B, 200 μM ATP was added to the wash in lanes 4 to 6). Lane 7: protein elution using 1 ml buffer containing 10 mM Tris–HCl pH 7.5, 100 mM NaH2PO4, 1 M imidazole, 1% Triton, 5 mM MgCl2, 2% sarcosyl. (C) Western blot of gel in panel B, detection was carried out using anti-phosphotyrosine antibody.
Mentions: Untagged Wzb and Wze were not retained by the nickel-charged affinity resin itself (data not shown), so they could be tested for affinity to resin-bound His6-Wzd. Wzb was not retained by His6-Wzd after washing the resin (data not shown), suggesting that no stable interaction was present between these two proteins. After incubating resin-bound His6-WzdC41 and Wze together, the resin was divided into two fractions for washing either with or without ATP. Wze was retained by His6-Wzd using the same washing conditions as with Wzb (Figure 4A). Without added ATP, the wash fraction did not contain any tyrosine-phosphorylated protein. However, when 200 μM ATP was added to the washing solution, Wze protein was released from resin-bound His6-Wzd in each of three successive washing steps (Figure 4B, lanes 4, 5 and 6). After eluting the remaining bound proteins from the same column, only a small quantity of non-phosphorylated Wze was associated with His6-Wzd (Figure 4B, Lane 7). Moreover, using the anti-phosphotyrosine antibody, the Wzd protein was revealed to be tyrosine-phosphorylated after washing with 200 μM ATP (Figure 4C).Figure 4

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