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N-wasp is essential for the negative regulation of B cell receptor signaling.

Liu C, Bai X, Wu J, Sharma S, Upadhyaya A, Dahlberg CI, Westerberg LS, Snapper SB, Zhao X, Song W - PLoS Biol. (2013)

Bottom Line: The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization.The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation.Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

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WASP and N-WASP negatively regulates each other.(A–D) TIRFM analysis of pWASP and pN-WASP in the contact zone of splenic B cells stimulated with membrane-tethered Fab′–anti-Ig (A and B). The MFI of pWASP (C) or pN-WASP (D) in the B-cell contact zone was quantified. (E–G) Flow cytometry analysis of the cellular MFI of pWASP or pN-WASP in splenic B cells from WKO and littermate control mice (F), and mouse splenic (E) and human PBMC B cells (G) that were treated with or without Wis and soluble mB-Fab′–anti-Ig plus streptavidin. (H) Flow cytometry analysis of the cellular MFI of pN-WASP in PBMC B cells from WAS patients and age-matched healthy donors that were incubated with or without soluble mB-Fab′–anti-Ig plus streptavidin for 2 min. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. *p<0.01, compared to B cells from wt or littermate control mice, without Wis treatment or healthy donors.
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pbio-1001704-g007: WASP and N-WASP negatively regulates each other.(A–D) TIRFM analysis of pWASP and pN-WASP in the contact zone of splenic B cells stimulated with membrane-tethered Fab′–anti-Ig (A and B). The MFI of pWASP (C) or pN-WASP (D) in the B-cell contact zone was quantified. (E–G) Flow cytometry analysis of the cellular MFI of pWASP or pN-WASP in splenic B cells from WKO and littermate control mice (F), and mouse splenic (E) and human PBMC B cells (G) that were treated with or without Wis and soluble mB-Fab′–anti-Ig plus streptavidin. (H) Flow cytometry analysis of the cellular MFI of pN-WASP in PBMC B cells from WAS patients and age-matched healthy donors that were incubated with or without soluble mB-Fab′–anti-Ig plus streptavidin for 2 min. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. *p<0.01, compared to B cells from wt or littermate control mice, without Wis treatment or healthy donors.

Mentions: The involvement of both WASP and N-WASP in BCR activation suggests a functional coordination between these two proteins. To understand their functional relationship, we compared the activation levels and kinetics of one of the two proteins in the presence and absence of the other using TIRFM and flow cytometry. Both the MFI of pWASP in the contact zone of cNKO B cells (Figure 7A,C) and the cellular MFI of wiskostatin-treated mouse B cells (Figure 7E) were increased compared to those in untreated control B cells. Conversely, the pN-WASP levels in the contact zone of WKO B cells (Figure 7B,D) and in WKO B cells (Figure 7F) were significantly higher than those in littermate control B cells. In both cases, the time taken for pN-WASP to peak was not changed. While the levels of pN-WASP and pWASP were changed, the overall protein levels of N-WASP and WASP did not change in WKO and cNKO B cells (Figure S5), indicating that the increased phosphorylation level is not due to an increase in protein expression. Consistent with the data from mouse models, treating human B cells from healthy subjects with the N-WASP inhibitor wiskostatin resulted in an increase in the cellular level of pWASP (Figure 7G). Furthermore, the level of pN-WASP was increased in PBMC B cells from WAS patients that did not express or expressed low levels of WASP, compared to that of healthy human controls (Figure 7H). These results indicate that WASP and N-WASP negatively regulate each other during BCR activation in both mouse and human B cells.


N-wasp is essential for the negative regulation of B cell receptor signaling.

Liu C, Bai X, Wu J, Sharma S, Upadhyaya A, Dahlberg CI, Westerberg LS, Snapper SB, Zhao X, Song W - PLoS Biol. (2013)

WASP and N-WASP negatively regulates each other.(A–D) TIRFM analysis of pWASP and pN-WASP in the contact zone of splenic B cells stimulated with membrane-tethered Fab′–anti-Ig (A and B). The MFI of pWASP (C) or pN-WASP (D) in the B-cell contact zone was quantified. (E–G) Flow cytometry analysis of the cellular MFI of pWASP or pN-WASP in splenic B cells from WKO and littermate control mice (F), and mouse splenic (E) and human PBMC B cells (G) that were treated with or without Wis and soluble mB-Fab′–anti-Ig plus streptavidin. (H) Flow cytometry analysis of the cellular MFI of pN-WASP in PBMC B cells from WAS patients and age-matched healthy donors that were incubated with or without soluble mB-Fab′–anti-Ig plus streptavidin for 2 min. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. *p<0.01, compared to B cells from wt or littermate control mice, without Wis treatment or healthy donors.
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pbio-1001704-g007: WASP and N-WASP negatively regulates each other.(A–D) TIRFM analysis of pWASP and pN-WASP in the contact zone of splenic B cells stimulated with membrane-tethered Fab′–anti-Ig (A and B). The MFI of pWASP (C) or pN-WASP (D) in the B-cell contact zone was quantified. (E–G) Flow cytometry analysis of the cellular MFI of pWASP or pN-WASP in splenic B cells from WKO and littermate control mice (F), and mouse splenic (E) and human PBMC B cells (G) that were treated with or without Wis and soluble mB-Fab′–anti-Ig plus streptavidin. (H) Flow cytometry analysis of the cellular MFI of pN-WASP in PBMC B cells from WAS patients and age-matched healthy donors that were incubated with or without soluble mB-Fab′–anti-Ig plus streptavidin for 2 min. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. *p<0.01, compared to B cells from wt or littermate control mice, without Wis treatment or healthy donors.
Mentions: The involvement of both WASP and N-WASP in BCR activation suggests a functional coordination between these two proteins. To understand their functional relationship, we compared the activation levels and kinetics of one of the two proteins in the presence and absence of the other using TIRFM and flow cytometry. Both the MFI of pWASP in the contact zone of cNKO B cells (Figure 7A,C) and the cellular MFI of wiskostatin-treated mouse B cells (Figure 7E) were increased compared to those in untreated control B cells. Conversely, the pN-WASP levels in the contact zone of WKO B cells (Figure 7B,D) and in WKO B cells (Figure 7F) were significantly higher than those in littermate control B cells. In both cases, the time taken for pN-WASP to peak was not changed. While the levels of pN-WASP and pWASP were changed, the overall protein levels of N-WASP and WASP did not change in WKO and cNKO B cells (Figure S5), indicating that the increased phosphorylation level is not due to an increase in protein expression. Consistent with the data from mouse models, treating human B cells from healthy subjects with the N-WASP inhibitor wiskostatin resulted in an increase in the cellular level of pWASP (Figure 7G). Furthermore, the level of pN-WASP was increased in PBMC B cells from WAS patients that did not express or expressed low levels of WASP, compared to that of healthy human controls (Figure 7H). These results indicate that WASP and N-WASP negatively regulate each other during BCR activation in both mouse and human B cells.

Bottom Line: The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization.The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation.Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

Show MeSH
Related in: MedlinePlus