Biomedical subjects

Paul L Stein

Publications and source records attributed to Paul L Stein.

7 recordsLinked to original sources

PubMed2006

Fyn kinase acts upstream of Shp2 and p38 mitogen-activated protein kinase to promote chemotaxis of mast cells towards stem cell factor.

The c-Kit receptor protein-tyrosine kinase plays a critical role in the differentiation, growth and survival of mast cells. Binding of its ligand stem cell factor (SCF), induces c-Kit dimerization, autophosphorylation, and recruitment of signaling proteins. The juxtamembrane sequence of c-Kit contains recruitment sites for the Src family kinases Fyn and Lyn, as well as Shp1 and Shp2 protein-tyrosine phosphatases. To characterize the role of Fyn in c-Kit signaling, we generated bone marrow-derived mast cells (BMMCs) from wild-type and Fyn knock-out mice. In contrast with previous studies of Lyn-deficient BMMCs, SCF treatment of Fyn-deficient BMMCs revealed no overt defects in the overall pattern of tyrosine phosphorylation, phosphatidylinositol 3' kinase recruitment to c-Kit, or phosphorylation of Stat3 transcription factor. However, Fyn-deficient mast cells showed a significant reduction in phosphorylation of Shp2 phosphatase and p38 mitogen-activated protein kinase. Defects in Shp2 and p38 phosphorylation were restored in Fyn-deficient mast cells transduced with a Fyn-expressing retrovirus (Fyn-rescue). Fyn-deficient BMMCs displayed reduced chemotaxis towards SCF, and this defect was corrected in Fyn-rescue cells. This study provides evidence that recruitment of both Shp2 and Fyn to juxtamembrane sites in c-Kit results in Shp2 phosphorylation, downstream signaling to p38 mitogen-activated protein kinase, and enhanced chemotaxis of mast cells.

Animals↗

PubMed2005

Regulation of NKT cell development by SAP, the protein defective in XLP.

The adaptor molecule SAP is expressed in T lymphocytes and natural killer (NK) cells, where it regulates cytokine production and cytotoxicity. Here, we show that SAP, encoded by the SH2D1A gene locus, also has a crucial role during the development of NKT cells, a lymphocyte subset with immunoregulatory functions in response to infection, cancer and autoimmune disease. Following stimulation with the NKT cell-specific agonist alpha-galactosyl ceramide (alphaGC), Sh2d1a-/- splenocytes did not produce cytokines or activate other lymphoid lineages in an NKT cell-dependent manner. While evaluating the abnormalities in alphaGC-induced immune responses, we observed that Sh2d1a-/- animals lacked NKT cells in the thymus and peripheral organs. The defect in NKT cell ontogeny was hematopoietic cell autonomous and could be rescued by reconstitution of SAP expression within Sh2d1a-/- bone marrow cells. Seventeen individuals with X-linked lymphoproliferative disease (XLP), who harbored germline mutations in SH2D1A, also lacked NKT cells. Furthermore, a female XLP carrier showed completely skewed X chromosome inactivation within NKT cells, but not T or B cells. Thus, SAP is a crucial regulator of NKT cell ontogeny in humans and in mice. The absence of NKT cells may contribute to the phenotypes of SAP deficiency, including abnormal antiviral and antitumor immunity and hypogammaglobulinemia.

Animals↗

PubMed2004

Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny.

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.

Animals↗

PubMed2004

Lineage choices in the developing thymus: choosing the T and NKT pathways.

Thymic development proceeds through several defined stages that generate not only alpha beta and gamma delta T cells but can produce dendritic cells and B cells. The earliest thymocytes exist in the CD4(-)CD8(-) double negative compartment within a heterogeneous fraction termed DN1. Recent progress has identified several candidate populations that may be the bone fide T-cell progenitor population. The potential roles of these populations, which include hematopoietic stem cells, early lymphocyte precursors, common lymphoid progenitors, and early T lineage progenitors are being elucidated. The alpha beta T-cell lineage consists of distinct subsets, one of which is NKT cells. The developmental relationship of NKT cells to conventional T cells has been controversial. Recent work has shown that these cells are probably derived from CD4(+)CD8(+) thymocytes. The discovery and application of CD1d tetramers has made it possible to more fully describe NKT-cell development.

Animals↗

PubMed2003

The mer receptor tyrosine kinase: expression and function suggest a role in innate immunity.

The mer receptor tyrosine kinase mediates phagocytosis of apoptotic cells and modulates cytokine production; it is also required for prevention of systemic autoimmune disease. Using a mer-specific antibody, we have confirmed the presence of mer on macrophages and now report its expression on NK cells, NKT cells, and dendritic cells (DC). We found that DC do not require mer for ingestion of apoptotic cells, as DC from mer-deficient mice phagocytose apoptotic cells normally. Mer was observed in splenic sections on cells outside follicular areas, probably representing DC and macrophages. Mer apparently participates in NKT-cell antigen-induced signaling, as NKT cells from mer-deficient mice evinced much lower cytokine production after in vivo alpha-galactosylceramide stimulation; this defect was intrinsic to the mer-deficient NKT cells. Taken together, these studies show mer expression on cells of the innate immune system. Mer, through its binding of lipid antigens, may not only mediate ingestion of apoptotic cells, but also signal events in NK cells, NKT cells, and DC.

Animals↗

PubMed2002

NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation.

NK T cells are a lymphocyte lineage that is selected by CD1d and is characterized by the ability to rapidly secrete large amounts of both IFN-gamma and IL-4 after TCR stimulation. Using reactivity to CD1d tetramers to define presumptive NK T cells, several NK T cell progenitor populations were characterized based upon NK marker expression and CD4 vs CD8 expression. The earliest populations were found to be negative for NK markers and could proliferate to IL-7, while mature NK T cells did not. The NK1.1(-) NK T cell progenitors were capable of up-regulating NK1.1 when transferred in vivo. Upon stimulation, the NK1.1(-) populations secrete IL-4, but little IFN-gamma. As the cells mature and up-regulate NK1.1, they acquire the ability to secrete IFN-gamma. Finally, the Tec family tyrosine kinase Itk is necessary for optimal NK1.1 up-regulation and hence final maturation of NK T cells. The itk(-/-) mice also display a progressive decrease in NK T cells in older animals, suggesting a further role in peripheral maintenance.

Animals↗

PubMed2001

'Srcasm: a novel Src activating and signaling molecule.

The Src family tyrosine kinase, Fyn, can facilitate regulation of cell proliferation and differentiation. Mice with mutations in the fyn gene have defects in the brain, immune system, and epidermal differentiation. To identify molecules that may interact with Fyn in the epidermis, we performed a yeast two-hybrid interaction screen of a murine keratinocyte library. A novel adaptor-like molecule was isolated and termed Srcasm for Src activating and signaling molecule. Murine Srcasm is a 52.7-kDa protein that contains a VHS membrane association domain and a number of tyrosine motifs suggesting that it may be a substrate for Src family kinases and serve as an adaptor protein. Northern blot analysis of murine tissues demonstrates that Srcasm expression is highest in brain and kidney. In situ hybridization analysis reveals that srcasm mRNA is expressed in regions of the epidermis and hair follicle where keratinocyte differentiation occurs. In the brain, srcasm mRNA distribution correlates with that of fyn, with both being highly expressed in the hippocampal and cerebellar Purkinje neurons. Fyn can phosphorylate Srcasm, and association of these molecules relies on cooperative binding between the SH2 and SH3 domains of Fyn and corresponding canonical binding sites in Srcasm. Srcasm is capable of interacting with Grb2 and the regulatory subunit of phosphoinositide 3-kinase, p85, in a phosphorylation-dependent manner. The evidence suggests that Srcasm may help promote Src family kinase signaling in cells.

Adaptor Proteins, Signal Transducing↗