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. 2010 Nov 2;107(44):18944-9.
doi: 10.1073/pnas.1007558107. Epub 2010 Oct 18.

SHEP1 Partners With CasL to Promote Marginal Zone B-cell Maturation

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Free PMC article

SHEP1 Partners With CasL to Promote Marginal Zone B-cell Maturation

Cecille D Browne et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The marginal zone is a cellular niche bordering the marginal sinus of the spleen that contains specialized B-cell and macrophage subsets poised to capture bloodborne antigens. Marginal zone B cells are retained in this niche by integrin-mediated signaling induced by G protein-coupled receptors (GPCRs) and, likely, the B-cell receptor (BCR). Sphingosine-1-phosphate (S1P) signaling via the S1P family of GPCRs is known to be essential for B-cell localization in the marginal zone, but little is known about the downstream signaling events involved. Here, we demonstrate that the adaptor protein SHEP1 is required for marginal zone B-cell maturation. SHEP1 functions in concert with the scaffolding protein CasL, because we show that SHEP1 and CasL are constitutively associated in B cells. SHEP1 association is required for the BCR or S1P receptor(s) to induce the conversion of CasL into its serine/threonine hyperphosphorylated form, which is important for lymphocyte adhesion and motility. Thus, SHEP1 orchestrates marginal zone B-cell movement and retention as a key downstream effector of the BCR and S1P receptors.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SHEP1-deficient mice have a disrupted splenic microarchitecture. (A) Splenocytes stained for B220, CD23, and CD21 and analyzed by flow cytometry (B220+ gated cells). The average frequencies of gated MZ B cells (CD23lo, CD21hi) are shown with SEM obtained from six mice. (B) Splenic sections stained for B220 (green), CD3 (blue), and MOMA-1 (red). The marginal zone is indicated by a white arrow. (C) Splenic sections stained for MARCO (blue), which detects MZ macrophages (dashed white arrow), and MOMA-1 (red), which detects metallophilic macrophages (solid white arrow).
Fig. 2.
Fig. 2.
B cell-specific SHEP1 deficiency leads to a reduced marginal zone B-cell compartment. (A) Lysates from splenic B cells and non-B cells from WT-CD19cre and SHEP1flox/flox-CD19cre mice immunoblotted for SHEP1 and actin. (B; Upper) WT-CD19cre and SHEP1flox/flox-CD19cre splenic B cells stained for CD23 and CD21. Frequencies of gated MZ B cells are shown with SDs obtained from three mice per group. (Lower) WT-CD19cre and SHEP1flox/flox-CD19cre splenic sections stained for B220 (green) and MOMA-1 (red). MZ is indicated by a white arrow. (C) WT-CD19cre and SHEP1flox/flox-CD19cre splenic sections stained for B220 (red) and MOMA-1 (green; Upper) and MARCO (blue) and MOMA-1 (red; Lower). (D) WT-CD19cre and SHEP1flox/flox-CD19cre splenic cells stained for B220, IgM, CD21, and CD23. The MZ+MZP compartments were gated as CD21hi, IgMhi (Upper Left) and subdivided into CD23hi (precursor MZB) and CD23lo (mature MZB) populations (Lower Left). The dot plot shows total cell numbers from five mice/group (Right). P values were calculated by using Student's t test.
Fig. 3.
Fig. 3.
Migration of SHEP1-deficient B cells is impaired in response to BLC/CXCL13 and S1P. (A) Migration of WT B cells in response to different S1P concentrations. The number of follicular (FB), MZ, and MZP B cells in the lower wells was divided by the corresponding number of each B-cell subpopulation in the input wells to obtain a measure of migration efficiency as a percentage of input cells. (B) Migration of WT-CD19cre and SHEP1flox/flox-CD19cre follicular (Left) and MZ+MZP (Right) B cells in response to 100 nM S1P with or without pertussis toxin (Ptx) preincubation. (C) Migration of splenic B cells from WT-CD19cre and SHEP1flox/floxCD19cre mice in response to 1 μg/mL BLC/CXCL13 for 4 h (Left). CXCR5 expression on splenic B cells (Right).
Fig. 4.
Fig. 4.
SHEP1 constitutively associates with CasL, and SHEP1 promotes CasL hyperphosphorylation. (A) Immunoprecipitated CasL from BAL17 cells stimulated with 1 μM S1P (Upper) or 10 μg/mL anti-IgM F(ab’)2 (Lower) and immunoblotted for SHEP1 and CasL. (B) Lysates from splenic B cells from WT-CD19cre and SHEP1flox/flox-CD19cre mice immunoblotted for CasL, SHEP1, and actin. (C) B-cell lysates incubated with or without λ-protein phosphatase (λ-PP) and immunoblotted for CasL and actin. (D) Immunoprecipitated CasL from splenic B cells from WT-CD19cre and SHEP1flox/flox-CD19cre mice stimulated with 10 μg/mL anti-IgM F(ab’)2 and immunoblotted for phosphoserine and SHEP1. Total lysates were immunoblotted for actin. (E) Lysates from splenic B cells from WT-CD19cre and SHEP1flox/flox-CD19cre mice were stimulated with 1 μM S1P and immunoblotted for CasL and actin.
Fig. 5.
Fig. 5.
Direct interaction between SHEP1 and CasL is required for hyperphosphorylation of CasL. (A) SHEP1flox/flox-CD19cre B cells were transduced with pMIT-SHEP1-WT or with pMIT-SHEP1-Y787E. Immunoprecipitated SHEP1 from sorted Thy1.1+ cells immunoblotted for CasL and SHEP1. (B) Lysates from Thy1.1+ SHEP1flox/flox-CD19cre B cells were transduced with pMIT, pMIT-SHEP1-WT, or pMIT-SHEP1-Y787E and immunoblotted for CasL, SHEP1, and actin.

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