T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70.
ABSTRACT The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.
[show abstract] [hide abstract]
ABSTRACT: Development, survival, and activation of B lymphocytes are controlled by signals emanating from the B-cell antigen receptor (BCR). The BCR has an autonomous signaling function also known as tonic signaling that allows for long-term survival of B cells in the immune system. Upon binding of antigen to the BCR, the tonic signal is amplified and diversified, leading to alteration in gene expression and B-cell activation. The spleen tyrosine kinase (Syk) intimately cooperates with the signaling subunits of the BCR and plays a central role in the amplification and diversification of BCR signals. In this review, we discuss the molecular mechanisms by which Syk activity is inhibited and activated at the BCR. Importantly, Syk acts not only as a kinase that phosphorylates downstream substrates but also as an adapter that can bind to a diverse set of signaling proteins. Depending on its interactions and localization, Syk can signal opposing cell fate decisions such as proliferation or differentiation of B cells.Immunological Reviews 11/2009; 232(1):286-99. · 11.15 Impact Factor
Article: Commensal microbiota alter the abundance and TCR responsiveness of splenic naïve CD4+ T lymphocytes.[show abstract] [hide abstract]
ABSTRACT: The epidemiologic risk of certain systemic immunologic diseases is affected by commensal or environmental microbiota, but the cellular basis of the "hygiene hypothesis" is poorly understood. In this study, we demonstrate that composition of the commensal microbiota affects the functional state of the peripheral naïve (CD62L(hi)CD44(lo)) T lymphocyte populations. Restricted flora (RF) mice (stably colonized with excess nonpathogenic Clostridium sp., and changes in other bacterial and fungal taxa) were distinguished after the neonatal period by a progressive deficiency in absolute numbers of naïve CD4+ and CD8+ T lymphocytes. SPF and RF mice had comparable levels of memory CD4+ and CD8+ T cells. This phenotype was attributable to the altered levels of certain commensals and their products, since germ-free mice had normal absolute numbers of splenic CD4+ and CD8+ T cells and their respective naïve and memory subsets. The naïve CD4+ T cell subset was functionally distinguished in RF mice versus SPF mice by TCR hyperresponsiveness, pro-inflammatory cytokine production, and increased activation-induced cell death. Biochemically, these traits were associated with higher basal phosphorylation of the TCR signaling proteins ZAP-70, Lck, and LAT. These findings indicate that enteric microbial products, through unknown cellular circuitry, influence steps in CD4 T cell differentiation moderating basal TCR signaling and immune responsiveness.Clinical Immunology 01/2006; 117(3):221-30. · 4.05 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors.BMC Cell Biology 02/2005; 6(1):20. · 2.59 Impact Factor