The SH3–SAM Adaptor HACS1 is Up-regulated in B Cell Activation Signaling Cascades

HACS1 is a Src homology 3 and sterile alpha motif domain–containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti–immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor–associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor κB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4–stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation

Cluster analysis of protein array results via similarity of Gene Ontology annotation

BACKGROUND: With the advent of high-throughput proteomic experiments such as arrays of purified proteins comes the need to analyse sets of proteins as an ensemble, as opposed to the traditional one-protein-at-a-time approach. Although there are several publicly available tools that facilitate the analysis of protein sets, they do not display integrated results in an easily-interpreted image or do not allow the user to specify the proteins to be analysed. RESULTS: We developed a novel computational approach to analyse the annotation of sets of molecules. As proof of principle, we analysed two sets of proteins identified in published protein array screens. The distance between any two proteins was measured as the graph similarity between their Gene Ontology (GO) annotations. These distances were then clustered to highlight subsets of proteins sharing related GO annotation. In the first set of proteins found to bind small molecule inhibitors of rapamycin, we identified three subsets containing four or five proteins each that may help to elucidate how rapamycin affects cell growth whereas the original authors chose only one novel protein from the array results for further study. In a set of phosphoinositide-binding proteins, we identified subsets of proteins associated with different intracellular structures that were not highlighted by the analysis performed in the original publication. CONCLUSION: By determining the distances between annotations, our methodology reveals trends and enrichment of proteins of particular functions within high-throughput datasets at a higher sensitivity than perusal of end-point annotations. In an era of increasingly complex datasets, such tools will help in the formulation of new, testable hypotheses from high-throughput experimental data

A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas

GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemotherapy resistance of human astrocytomas

Functional Cooperation between c-Cbl and Src-Like Adaptor Protein 2 in the Negative Regulation of T-Cell Receptor Signaling

Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface

The Cell Fate Determinant Numb Interacts with EHD/Rme-1 Family Proteins and Has a Role in Endocytic Recycling

The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and α-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors