Immunoinhibitory adapter protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin dynamics and B cell spreading.

Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

SLy2-deficiency promotes B-1 cell immunity and triggers enhanced production of IgM and IgG2 antibodies against pneumococcal vaccine.

BACKGROUND: Despite the benefits of existing vaccines, Streptococcus pneumoniae is still responsible for the greatest proportion of respiratory tract infections around the globe, thereby substantially contributing to morbidity and mortality in humans. B-1 cells are key players of bacterial clearance during pneumococcal infection and even provide long-lasting immunity towards S. pneumoniae. Previous reports strongly suggest an essential role of the immunoinhibitory adapter Src homology domain 3 lymphocyte protein 2 (SLy2) for B-1 cell-mediated antibody production. The objective of this study is to evaluate S. pneumoniae-directed B cell responses in the context of SLy2 deficiency. METHODS: B-1 cell populations were analyzed via flow cytometry before and after pneumococcal immunization of SLy2-deficient and wild-type control mice. Global and vaccine-specific immunoglobulin M (IgM) and IgG antibody titers were assessed by enzyme-linked immunosorbent assay. To investigate survival rates during acute pneumococcal lung infection, mice were intranasally challenged with S. pneumoniae (serotype 3). Complementary isolated splenic B cells were stimulated in vitro and their proliferative response was assessed by fluorescent staining. In vitro antibody secretion was quantified by LEGENDplex. RESULTS: We demonstrate increased frequencies of B-1 cells and elevated titers of preantigenic IgM in SLy2-deficient mice. In addition, these mice produce significantly more amounts of IgM and IgG2 upon pneumococcal vaccination. Knocking out SLy2 did not induce survival advantages in our murine model of acute pneumonia, indicating the presence of compensatory mechanisms. CONCLUSION: Our results reveal reinforced specific antibody responses towards pneumococcal polysaccharides and enhanced IgG2 secretion as a consequence of SLy2 deficiency, which could be relevant to the development of more efficient vaccines.

The catalytic PI3K isoforms p110gamma and p110delta contribute to B cell development and maintenance, transformation, and proliferation.

Class I PI3K-dependent signaling regulates cell proliferation, differentiation, and survival. Analysis of gene-deficient mice revealed specific roles for the hematopoietically expressed PI3K catalytic subunits, p110gamma and p110delta, in development and function of T and B lymphocytes. However, the functional redundancy between these two PI3K isoforms in the B cell lineage remains unclear. Here, we demonstrate that p110delta and p110gamma are expressed in B cells at early developmental stages. Normal B cell differentiation requires both isoforms, as p110gamma/p110delta double deficiency causes an increased percentage of CD43(hi)/B220(+)/CD19(-) cells as compared with single deficiency. Interestingly, initial transformation efficiency of B cell precursors was strongly reduced in double-deficient cells following transformation by p185 bcr-abl or v-abl oncogenes as compared with single-deficient cells. The requirement of p110gamma and p110delta in B cell development is underlined by reduced splenic B cell numbers of p110gamma/p110delta double-deficient mice and of lethally irradiated wild-type mice reconstituted with double-deficient BM. Moreover, the peripheral maintenance of p110gamma/p110delta double-deficient T and B cells was highly impaired following adoptive transfer of double-deficient splenocytes into wild-type mice. Functionally, LPS stimulation of splenocytes revealed proliferation defects resulting in decreased survival of p110gamma/p110delta double-deficient B cells, which correlated with impaired induction of D-type cyclins and Bcl-X(L). Surprisingly, this was not observed when purified B cells were analyzed, indicating a contribution of likely cell-extrinsic factor(s) to the impaired proliferation of double-deficient B cells. Thus, we provide novel evidence that p110gamma and p110delta have overlapping and cell-extrinsic roles in the development, peripheral maintenance, and function of B cells.

Isoform-selective interaction of the adaptor protein Tks5/FISH with Sos1 and dynamins.

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.