Genetic and Biochemical Approaches to Tyrosine Kinase Function

Human Genome Research

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Abstract

Summary: Our laboratory studies signal transduction in the immune system involving tyrosine phosphorylation and non-receptor tyrosine kinases, molecules required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth and development involved in cancer. Mutations affecting such signaling pathways have been found to give rise to multiple human disorders including several primary immunodeficiencies. Our work is focused on three sets of molecules implicated in immunodeficiencies: the Tec kinases, the Wiskott-alrich Syndrome Protein and SAP. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how these molecules contribute to normal function of cells in the immune system and their abnormal function in immunodeficiencies and autoimmune disorders. Our goals include understanding how manipulation of these pathways can help in the understanding of diseases of the immune system and in the development of therapeutics for immune-mediated disease, as well as responses to infection. [unreadable] [unreadable] A large portion of our work focuses on the Tec family of tyrosine kinases and their functions in T lymphocytes. Mutations affecting the prototypical member of this family, Btk, are responsible for the genetic disorder X-linked agammmaglobulimemia, which affects B cell development and function. We have shown that mutation of Tec family kinases expressed in T cells also severely impairs T lymphocyte function in mice and influence responses to infections in vivo. We have further defined that, in addition to their recognized roles in antigen receptor induced activation of PLC-gamma, a key enzyme required for Ca++ mobilization, the Tec kinases play an important role in regulation of the actin cytoskeleton and cell adhesion in T lymphocytes. Our work argues that cytoskeletal defects may contribute to the phenotypes associated with Tec kinase deficiency, findings that have now been extended by others to phenotypes seen in X-linked agammaglobulinemia.[unreadable] [unreadable] In continuing work, we are also examining the effects of mutation of Tec kinases on T cell development and differentiation in vivo. We have recently found that mutation of the Tec kinase Itk affects development of conventional T lymphocytes and that most of the CD8 cells that develop in these mice resemble a normally rare population of innate-like T cells that are important for immediate early responses to bacterial infections. Our work places the Tec kinases as part of pathways regulating the differentiation of conventional and innate-type lymphocytes. We have further examined the role of Tec kinases in CD4 T helper cell differentiation. Previous work from our laboratory and others has demonstrated that the Tec kinase Itk is required to TH2 immune responses, the type of immune response that drives asthma and hypersensitivity. Work from others have implicated the Tec kinase Rlk in driving TH1 responses, a type of response associated with inflammation and autoimmunity. By overexpressing Rlk in Itk-deficient animals, we recently found that Rlk can substitute for Itk in TH2 responses when overexpressed, suggesting that their functions in different immune responses may be secondary to their patterns of expression, rather than distinct functional responses.[unreadable] [unreadable] Productive infection of T cells by the Human Immunodeficiency Virus (HIV) requires full T cell activation as well as chemokine receptors that serve as co-receptors along with CD4 for HIV entry into cells. We have previously found that the Tec kinase Itk is required for full TCR-induced activation of T cells, as well as for actin cytoskeletal reorganization in response to both TCR and chemokine stimulation. Intriguingly, HIV infection also requires actin reorganization in response to the viral envelope protein, gp120 (which binds both CD4, a part of the TCR complex, and the chemokine receptors CXCR4 and CCR5). We hypothesized that Itk may influence HIV infection. Using siRNA directed against Itk, a dominant-negative Itk mutant and a chemical inhibitor of Itk, synthesized by Craig Thomas, we have demonstrated that blocking Itk expression or function can inhibit HIV infection by affecting multiple stages of the HIV life-cycle including viral entry, transcription of HIV, and viral production/egress.[unreadable] [unreadable] As an extension of our studies on T lymphocyte signal transduction, we are examining other signaling molecules involved in primary immunodeficiencies and their effects on T cell differentiation and function. The Wiskott-Aldrich Syndrome Protein (WASP) is another actin regulatory protein expressed in hematopoietic cells that is regulated by pathways involving the Tec kinases; mutations affecting WASP lead to a primary immunodeficiency associated with defective T cell actin cytoskeleton and impaired activation and cytokine production in vitro. However, in addition to increased susceptibility to infections, a high proportion of patients with WAS also develop autoimmune disorders. It has been a mystery as to why these patients with impaired T cell function show evidence of increased T cell-mediated pathology in autoimmunity. We and three other groups have found that WASP-deficiency leads to impaired function of regulatory T cells, an important negative regulatory component of the immune system, perhaps accounting for the increased autoimmunity seen in WAS. We are currently investigating the consequences of these observations for immune responses in vivo.[unreadable] [unreadable] The third major project in our laboratory is the evaluation of SAP, a small SH2 containing adaptor protein that binds tyrosine phosphorylated proteins. Mutations affecting SAP are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of a series of immunomodulatory receptors related to SLAM. We had previously generated SAP-deficient mice and found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. In the last year, we have published that SAP also plays a critical role in responses to immunization. We further showed that the impaired antibody responses in these mice is secondary to a defect in CD4+ T cells, ie SAP deficient T cells fail to provide an essential signal to B cells for generating long-term antibody responses, a critical step for the development of successful immunization and vaccine development. To understand the defect in T cells in the SAP-deficient mice, we have continued to examine T cell function and biochemistry of T cell activation in cells from SAP-deficient mice. We have previously shown that T cells from SAP deficient mice show dramatic defects in TH2 cytokine production (IL-4, 5, and 10) in response to TCR stimulation. However, in a report last year, we showed that their defects in T cell help for antibody production can be separated from their TH2 defects. Moreover, while rescue of the defects in IL-4 production in SAP-deficient cells requires recruitment of the Fyn tyrosine kinase, rescue of the humoral defects does not require a SAP-Fyn interaction and may involve distinct signaling pathways. Our current work has focused on biochemical pathways downstream of SAP to help decipher the signaling pathways that cause the impaired antibody production, as well as intravital microscopy to evaluate how these biochemical defects affect T:B cell interaction in vivo and in vitro. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder. Moreover, understanding the cellular interactions and signals that are defective in these mice is of high importance for understanding the requirements for successful vaccine development.