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Unc119 protects from Shigella infection by inhibiting the Abl family kinases.

Vepachedu R, Karim Z, Patel O, Goplen N, Alam R - PLoS ONE (2009)

Bottom Line: We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis.Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality.Unc119 is an inducible protein.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO, USA.

ABSTRACT

Background: Bacteria engage cell surface receptors and intracellular signaling molecules to enter the cell. Unc119 is an adaptor protein, which interacts with receptors and tyrosine kinases. Its role in bacterial invasion of cells is unknown.

Methodology/principal findings: We used biochemical, molecular and cell biology approaches to identify the binding partners of Unc119, and to study the effect of Unc119 on Abl family kinases and Shigella infection. We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis. Unc119 interacts with Abl family kinases and inhibits their kinase activity. As a consequence, it inhibits Crk phosphorylation, which is essential for Shigella infection. Unc119 co-localizes with Crk and Shigella in infected cells. Shigella infectivity increases in Unc119-deficient epithelial and macrophage cells. In a mouse model of shigellosis cell-permeable TAT-Unc119 inhibits Shigella infection. Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality. Unc119 is an inducible protein. Its expression is upregulated by probacteria and bacterial products such as lipopolysacharide and sodium butyrate. The latter inhibits Shigella infection in mouse lungs but is ineffective in Unc119 deficiency.

Conclusions: Unc119 inhibits signaling pathways that are used by Shigella to enter the cell. As a consequence it provides partial but significant protection from Shigella infections. Unc119 induction in vivo boosts host defense against infections.

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Related in: MedlinePlus

Unc119 blocks Shigella infection by inhibiting Abl family kinases.(A) Unc119 inhibits Abl and Arg kinase activity. Abl and Arg kinases were immunoprecipitated from the 3T3 cells and their kinase activity was measured by incorporation of 32P-γ-ATP into two different substrates- GST-Crk (left top two panels) and GST-Abltide (right top two panels) in presence of increasing concentrations of recombinant Unc119 (Top and middle panels) or Unc119-derived SH2 and SH3 motif peptides (bottom panels. The samples were then separated by SDS-PAGE, transferred to PVDF membrane, and exposed to storage phosphor screen. The phosphorylation was captured using the Typhoon Variable Mode Imager. The membranes were reprobed for the kinases to show equal loading (lower panels, N = 3). (B) Unc119 overexpression inhibits actin polymerization. 3T3 cells overexpressing Unc119-GFP (left two panels) or GFP (right panels) were stained for actin by phalloidin (left two panels and right bottom panel. The upper left panel is a higher magnification of three cells from the bottom left image (bottom right corner). Arrows indicate cells overexpressing Unc119-GFP (N = 3). (C) Unc119 knockdown has no effect on Shigella infection in the absence of Abl family kinase activity. 3T3, Abl−/−, Arg−/− and Abl−/−Arg−/− cells were transfected with siControl and siUnc119 RNA. After 48 h the cells were infected with Shigella and the infectivity was measured. The data represents mean±SD of 3 independent experiments performed in triplicates (*P<0.038).
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pone-0005211-g004: Unc119 blocks Shigella infection by inhibiting Abl family kinases.(A) Unc119 inhibits Abl and Arg kinase activity. Abl and Arg kinases were immunoprecipitated from the 3T3 cells and their kinase activity was measured by incorporation of 32P-γ-ATP into two different substrates- GST-Crk (left top two panels) and GST-Abltide (right top two panels) in presence of increasing concentrations of recombinant Unc119 (Top and middle panels) or Unc119-derived SH2 and SH3 motif peptides (bottom panels. The samples were then separated by SDS-PAGE, transferred to PVDF membrane, and exposed to storage phosphor screen. The phosphorylation was captured using the Typhoon Variable Mode Imager. The membranes were reprobed for the kinases to show equal loading (lower panels, N = 3). (B) Unc119 overexpression inhibits actin polymerization. 3T3 cells overexpressing Unc119-GFP (left two panels) or GFP (right panels) were stained for actin by phalloidin (left two panels and right bottom panel. The upper left panel is a higher magnification of three cells from the bottom left image (bottom right corner). Arrows indicate cells overexpressing Unc119-GFP (N = 3). (C) Unc119 knockdown has no effect on Shigella infection in the absence of Abl family kinase activity. 3T3, Abl−/−, Arg−/− and Abl−/−Arg−/− cells were transfected with siControl and siUnc119 RNA. After 48 h the cells were infected with Shigella and the infectivity was measured. The data represents mean±SD of 3 independent experiments performed in triplicates (*P<0.038).

Mentions: As mentioned previously, Unc119 activates the Src family of tyrosine kinase. Since Abl and Arg kinases belong to a closely related tyrosine kinase family, we asked if Unc119 affected the kinase activity of Abl and Arg. We examined the effect of recombinant Unc119 on the enzymatic activity of Abl family kinases in an in vitro kinase assay using GST-Crk as a substrate. Unc119 inhibited the phosphorylation of GST-Crk in a dose-dependent manner (Figure 4A, left panels). The inhibitory effect was stronger on Arg than Abl kinase. Since Unc119 directly interacts with Crk through its SH3 domain, it is possible Unc119 sequesters Crk and thereby prevents its phosphorylation without affecting Abl kinase activity. To address this concern we used a different substrate that lacks the Crk SH3 domain. We used GST-Abltide, which is a fusion peptide containing the Abl phosphorylation motif (EAIYAAPFAKKK) with an N-terminal GST-tag. The inhibition of phosphorylation of GST-Abltide by Unc119 (Fig. 4A, right panels) was actually stronger than that of GST-Crk. The results suggest that the inhibitory effect of Unc119 on Abl kinases is direct. To demonstrate the region of Unc119 that interacts with the kinases and inhibits the enzymatic activity we used peptide sequences of the SH2-binding motif (TCEHIYDFPPLS) and SH3-binding motif (QGKQPIGPED) from Unc119 in the kinase assay. The presence of the SH2 binding motif had no effect on the kinase activity (Figure 4A, lower panels). On the other hand, the SH3-binding motif of Unc119 caused inhibition of GST-Crk phosphorylation indicating that an SH3-mediated interaction is responsible for inhibition of the enzymatic activity.


Unc119 protects from Shigella infection by inhibiting the Abl family kinases.

Vepachedu R, Karim Z, Patel O, Goplen N, Alam R - PLoS ONE (2009)

Unc119 blocks Shigella infection by inhibiting Abl family kinases.(A) Unc119 inhibits Abl and Arg kinase activity. Abl and Arg kinases were immunoprecipitated from the 3T3 cells and their kinase activity was measured by incorporation of 32P-γ-ATP into two different substrates- GST-Crk (left top two panels) and GST-Abltide (right top two panels) in presence of increasing concentrations of recombinant Unc119 (Top and middle panels) or Unc119-derived SH2 and SH3 motif peptides (bottom panels. The samples were then separated by SDS-PAGE, transferred to PVDF membrane, and exposed to storage phosphor screen. The phosphorylation was captured using the Typhoon Variable Mode Imager. The membranes were reprobed for the kinases to show equal loading (lower panels, N = 3). (B) Unc119 overexpression inhibits actin polymerization. 3T3 cells overexpressing Unc119-GFP (left two panels) or GFP (right panels) were stained for actin by phalloidin (left two panels and right bottom panel. The upper left panel is a higher magnification of three cells from the bottom left image (bottom right corner). Arrows indicate cells overexpressing Unc119-GFP (N = 3). (C) Unc119 knockdown has no effect on Shigella infection in the absence of Abl family kinase activity. 3T3, Abl−/−, Arg−/− and Abl−/−Arg−/− cells were transfected with siControl and siUnc119 RNA. After 48 h the cells were infected with Shigella and the infectivity was measured. The data represents mean±SD of 3 independent experiments performed in triplicates (*P<0.038).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2667249&req=5

pone-0005211-g004: Unc119 blocks Shigella infection by inhibiting Abl family kinases.(A) Unc119 inhibits Abl and Arg kinase activity. Abl and Arg kinases were immunoprecipitated from the 3T3 cells and their kinase activity was measured by incorporation of 32P-γ-ATP into two different substrates- GST-Crk (left top two panels) and GST-Abltide (right top two panels) in presence of increasing concentrations of recombinant Unc119 (Top and middle panels) or Unc119-derived SH2 and SH3 motif peptides (bottom panels. The samples were then separated by SDS-PAGE, transferred to PVDF membrane, and exposed to storage phosphor screen. The phosphorylation was captured using the Typhoon Variable Mode Imager. The membranes were reprobed for the kinases to show equal loading (lower panels, N = 3). (B) Unc119 overexpression inhibits actin polymerization. 3T3 cells overexpressing Unc119-GFP (left two panels) or GFP (right panels) were stained for actin by phalloidin (left two panels and right bottom panel. The upper left panel is a higher magnification of three cells from the bottom left image (bottom right corner). Arrows indicate cells overexpressing Unc119-GFP (N = 3). (C) Unc119 knockdown has no effect on Shigella infection in the absence of Abl family kinase activity. 3T3, Abl−/−, Arg−/− and Abl−/−Arg−/− cells were transfected with siControl and siUnc119 RNA. After 48 h the cells were infected with Shigella and the infectivity was measured. The data represents mean±SD of 3 independent experiments performed in triplicates (*P<0.038).
Mentions: As mentioned previously, Unc119 activates the Src family of tyrosine kinase. Since Abl and Arg kinases belong to a closely related tyrosine kinase family, we asked if Unc119 affected the kinase activity of Abl and Arg. We examined the effect of recombinant Unc119 on the enzymatic activity of Abl family kinases in an in vitro kinase assay using GST-Crk as a substrate. Unc119 inhibited the phosphorylation of GST-Crk in a dose-dependent manner (Figure 4A, left panels). The inhibitory effect was stronger on Arg than Abl kinase. Since Unc119 directly interacts with Crk through its SH3 domain, it is possible Unc119 sequesters Crk and thereby prevents its phosphorylation without affecting Abl kinase activity. To address this concern we used a different substrate that lacks the Crk SH3 domain. We used GST-Abltide, which is a fusion peptide containing the Abl phosphorylation motif (EAIYAAPFAKKK) with an N-terminal GST-tag. The inhibition of phosphorylation of GST-Abltide by Unc119 (Fig. 4A, right panels) was actually stronger than that of GST-Crk. The results suggest that the inhibitory effect of Unc119 on Abl kinases is direct. To demonstrate the region of Unc119 that interacts with the kinases and inhibits the enzymatic activity we used peptide sequences of the SH2-binding motif (TCEHIYDFPPLS) and SH3-binding motif (QGKQPIGPED) from Unc119 in the kinase assay. The presence of the SH2 binding motif had no effect on the kinase activity (Figure 4A, lower panels). On the other hand, the SH3-binding motif of Unc119 caused inhibition of GST-Crk phosphorylation indicating that an SH3-mediated interaction is responsible for inhibition of the enzymatic activity.

Bottom Line: We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis.Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality.Unc119 is an inducible protein.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO, USA.

ABSTRACT

Background: Bacteria engage cell surface receptors and intracellular signaling molecules to enter the cell. Unc119 is an adaptor protein, which interacts with receptors and tyrosine kinases. Its role in bacterial invasion of cells is unknown.

Methodology/principal findings: We used biochemical, molecular and cell biology approaches to identify the binding partners of Unc119, and to study the effect of Unc119 on Abl family kinases and Shigella infection. We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis. Unc119 interacts with Abl family kinases and inhibits their kinase activity. As a consequence, it inhibits Crk phosphorylation, which is essential for Shigella infection. Unc119 co-localizes with Crk and Shigella in infected cells. Shigella infectivity increases in Unc119-deficient epithelial and macrophage cells. In a mouse model of shigellosis cell-permeable TAT-Unc119 inhibits Shigella infection. Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality. Unc119 is an inducible protein. Its expression is upregulated by probacteria and bacterial products such as lipopolysacharide and sodium butyrate. The latter inhibits Shigella infection in mouse lungs but is ineffective in Unc119 deficiency.

Conclusions: Unc119 inhibits signaling pathways that are used by Shigella to enter the cell. As a consequence it provides partial but significant protection from Shigella infections. Unc119 induction in vivo boosts host defense against infections.

Show MeSH
Related in: MedlinePlus