Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity.

E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.

Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo.

Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3-kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65(PI3K), developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phenotype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65(PI3K)-induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65(PI3K) Tg p53(-/-) mice indicated that PI3K contributes to tumor development. These observations demonstrate that constitutive activation of PI3K extends T cell survival in vivo, affects T cell homeostasis, and contributes to tumor generation, supporting a model in which selective increases in one type of PTEN substrate, the PI3K-derived lipid products, induce tumorigenesis. PI3K thus emerges as a potential target in autoimmune disease and cancer therapy.

Evidence for B cell participation in the in vitro and in vivo maintenance of in vivo staphylococcal enterotoxin B-induced T cell anergy.

The mechanisms involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy are poorly understood. Here, we demonstrate that CD4+ T cell anergy induced by SEB treatment is under partial B cell control. This effect is not mediated by anti-SEB antibodies or any in vitro B cell-produced suppresser factor. At day 13 after SEB immunization, T cells from B cell-deficient mice proliferate upon in vitro stimulation with SEB. These results suggest that SEB-induced T cell anergy is reversible and that B cells have an important function in anergy maintenance in CD4+ T cells, both in vivo and in vitro.

The catalytic domain of pp56(lck), but not its regulatory domain, is sufficient for inducing IL-2 production.

The lymphoid src kinase pp56(lck) has been shown to be essential for the induction of different T lymphocyte responses, including CD4-mediated enhancement of Ag-induced T cell activation, early T cell differentiation, induction of IL-2 production, and cytotoxicity. It is assumed that pp56(lck) acts on these processes by phosphorylating substrates. However, it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function. In this report we address the contribution of the regulatory and catalytic domains of pp56(lck) to another function of this enzyme independent of CD4: TCR-induced IL-2 production. Two pp56(lck) mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated, and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells (NF-AT) region of the IL-2 promoter were compared. Only the catalytic, but not the NH2 regulatory, domain of pp56(lck) was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response. Moreover, the catalytic domain of pp56(lck) was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56(lck). We conclude that different domains of the pp56(lck) molecule contribute to regulate distinct biologic functions. In fact, while the NH2 regulatory domain is sufficient to mediate CD4 accessory function, we show here that the catalytic domain of pp56(lck) is sufficient for induction of IL-2 production, mimicking TCR ligation.

Role of the autophosphorylation site on the biological function of pp56lck.

Src-family tyrosine kinases act as signaling molecules in a wide array of cellular activation processes. The existence of the various src-family members reflects the requirement for different cell-surface receptors to transmit cell-type specific intracellular signals. The structural basis for the functional specificity of src-kinases is being actively investigated. In the present report we have analysed the contribution of the area surrounding the autophosphorylation site (located at subdomain VII of the catalytic domain) in determining src-kinases activity and functional specificity. To this end we analysed the kinase activities of the lymphoid src-kinase pp56lck and a mutant of pp56lck in which this region has been exchanged for the corresponding area of the serine/threonine kinase c-Raf. Our studies indicate that the change at subdomain VII affected the ability of pp56lck to phosphorylate physiological substrates. Furthermore, when analysed in T cells, the mutant at subdomain VII failed to induce interleukin-2 production, a specific biological function of pp56lck. Thus, the area surrounding the autophosphorylation site of pp56lck plays a critical role in mediating its specific biological function.

Lck unique domain influences Lck specificity and biological function.

Src-family tyrosine kinases share structural and amino acid sequence homology, particularly in the catalytic domain as well as in the SH2 and SH3 domains of the regulatory region. However, each src-family member also contains a unique domain which is specific to and characteristic of each individual tyrosine kinase. These unique or specific domains may contribute to the functional specificity of each src-family kinase. To address this possibility, we analyzed the kinase activities and substrate specificities of the lymphoid src-kinase, pp56lck, and a mutant of pp56lck lacking its specific domain. Our data show that both the wild type enzyme and the specific domain-deleted mutant displayed similar affinities for ATP and the non-physiological substrate denatured enolase. However, the specific domain-deleted mutant failed to phosphorylate a number of physiological substrates of pp56lck. In addition, the ability of pp56lck to mediate induction of the interleukin-2 promoter was strongly impaired upon deletion of its specific domain. Thus, the unique domain is not required for the intrinsic kinase activity of pp56lck, however, it influences substrate preference and contributes to the unique physiological function of this src-family tyrosine kinase.

Tyrosine kinase specific motif at subdomain VIII does not confer specificity for tyrosine.

The majority of protein kinases fall within one of the two broad classes, kinases that phosphorylate serine or threonine and kinases that phosphorylate tyrosine. The structural basis that confers residue specificity is not known. However, it has been hypothesized that a region in subdomain VIII of the catalytic domain may be involved in determining kinase specificity. This region contains a motif which is conserved among serine/threonine kinases and different from the one conserved among tyrosine kinases. We have prepared a chimera of the tyrosine kinase pp56lck in which the tyrosine kinase motif at subdomain VIII has been exchanged for the corresponding region of the serine/threonine kinase c-Raf. Our results indicate that this motif itself does not confer amino acid specificity since the chimeric kinase still displays specificity for tyrosine.