Roger M. Perlmutter

A Syndrome of Multiorgan Hyperplasia with Features of Gigantism, Tumorigenesis, and Female Sterility in p27Kip1-Deficient Mice

SUMMARY Targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T lymphocyte proliferation, and T cells showed enhanced IL-2 responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia, suggesting that p27 and Rb function in the same regulatory pathway. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of secondary ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.

Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs

p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins.

Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn

Considerable evidence supports the hypothesis that the nonreceptor protein tyrosine kinase p59fyn participates in signal transduction from the T cell receptor (TCR). To examine this hypothesis in detail, we have produced mice that lack the thymic isoform of p59fyn but retain expression of the brain isoform of the protein. fynTnull mice exhibit a remarkably specific lymphoid defect: thymocytes are refractile to stimulation through the TCR with mitogen or antigen, while peripheral T cells, following what appears to be a normal maturation sequence, reacquire significant signaling capabilities. These data confirm that p59fynT plays a pivotal role in TCR signal transduction and demonstrate that additional developmentally regulated signaling components also contribute to TCR-induced lymphocyte activation.

Three distinct IL-2 signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic cell proliferation

Two interleukin-2 receptor-dependent signaling pathways have thus far been identified: the c-fos/c-jun induction pathway mediated by src family protein-tyrosine kinases and the c-myc induction pathway. Here, we provide evidence for the existence of a third, rapamycin-sensitive pathway, which results in the induction of another proto-oncogene, bcl-2. In the hematopoietic cell line BAF-B03, the expression of any two of lckF505 (an active form of p56lck), Bcl-2, or c-Myc is sufficient to promote transit of the cell cycle, regardless of the activation state of the third pathway. We also provide evidence that epidermal growth factor receptor signaling may act through the same pathway that involves p56lck. These studies demonstrate a novel approach to dissecting signaling pathways regulating cellular proliferation.

A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA

Protein-tyrosine kinases are implicated in the control of normal and neoplastic cell growth. We have used molecular cloning strategies to characterize a lymphocyte-specific protein-tyrosine kinase gene distinct from but closely related to src and yes. This gene, encoded by a genetic locus defined here as lskT, is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Thus alterations in the structure or expression of this protein-tyrosine kinase gene may in some cases mediate neoplastic transformation. In addition, transcription of the normal lskT gene is restricted to cells of lymphoid origin. We infer that the lskT-encoded protein-tyrosine kinase may aid in transducing proliferative or differentiative signals unique to lymphocytes.

A dominant-negative transgene defines a role for p56lck in thymopoiesis.

The lymphocyte‐specific protein tyrosine kinase p56lck participates in T cell signaling through functional interactions with components of the T cell antigen receptor complex and the interleukin‐2 receptor. Additional insight into the function of p56lck has now been obtained through the generation of transgenic animals expressing high levels of a catalytically inactive form of this kinase (p56lckR273). Mice bearing the lckR273 transgene manifested a severe defect in the production of virtually all T lymphocytes. Those exceptional CD3+ cells that escaped the effects of the lckR273 transgene were confined primarily to the T cell subset that expresses gamma/delta T cell receptors. Remarkably, construction of a dose‐response curve for the effects of the lckR273 transgene revealed that developmental arrest of thymocytes occurred at a discrete stage in the normal T cell maturation pathway, corresponding to a point at which thymoblasts ordinarily begin a series of mitotic divisions that result in expansion and maturation. These results suggest that p56lck normally regulates T cell production by metering the replicative potential of immature thymoblasts.

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant‐negative p21ras protein (H‐rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck‐H‐rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR‐derived signals in mature CD4+8‐ or CD8+4‐ thymocytes. In contrast, some TCR‐derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant‐negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H‐Y‐specific TCR and lck‐H‐rasN17 demonstrated that antigen‐specific negative selection occurs normally in the presence of p21H‐rasN17. Superantigen‐induced negative selection in vivo also proceeded unhindered in H‐rasN17 thymocytes. In contrast, positive selection of thymocytes in the H‐Y mice was severely compromised by the presence of p21H‐rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase

Stimulation of the T lymphocyte antigen receptor‐CD3 complex (TCR‐CD3) causes T cell activation by a process associated with increased phosphatidylinositol‐specific phospholipase C (PI‐PLC) activity. Evidence exists suggesting that GTP‐binding (G) proteins, particularly the pertussis toxin (PT)‐sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR‐CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte‐specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL‐2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck‐PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT‐sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

The impact of systems approaches on biological problems in drug discovery

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 10 OCTOBER 2004 1215 The production of new molecular entities (NMEs) endowed with salutary medicinal properties provides a key metric by which the effectiveness of the pharmaceutical industry may be judged. Nearly a decade ago, Jürgen Drews1 noted that the combined output of NMEs by all major pharmaceutical companies at the time was insufficient to support the healthy growth of the group. Indeed, a recent analysis documents a 30-year decline in pharmaceutical R&D productivity2. This decline in productivity has occurred despite accelerating investment in biomedical research on the part of both industry and governments, with compound annual growth rates in research expenditures approaching 13% (ref. 2). Approvals aside, the number of late-stage clinical trials supported by the pharmaceutical industry has also declined during the past decade3, auguring poorly for future triumphs in disease interdiction. Despite several notable discoveries, the emergence of major biotech companies has done little to improve the yield of new disease-ameliorating molecules. How is it that extraordinary advances in biomedical research over the past decade have exerted so little positive effect on drug discovery? We argue here that an inability to visualize the complexity of biological systems has impeded the identification of novel therapies. In the future, application of systems approaches to drug discovery promises to have a profound impact on medical practice, permitting a comprehensive evaluation of underlying predisposition to disease, disease diagnosis and disease progression. In the near term, systems biology will provide powerful means for validating new drug targets, improving the success with which pharmaceuticals are identified. Farther into the future, the same approaches will drive the development of early diagnostics, enabling disease stratification, individualized therapy and ultimately preventive drugs, based on both genetic and environmental considerations. Although systems biology as currently envisioned does not have a direct impact on the chemistry of identifying drugs or pharmacological challenges of drug metabolism, it may provide rapid and useful assays for these in the future.

Fyn can partially substitute for Lck in T lymphocyte development.

Lck, a Src family tyrosine kinase, transduces signals important for the development of alphabeta and gammadelta T cells. However, T cell development is only partially compromised in Lck-deficient mice, suggesting that other kinases may also transduce pre-TCR or TCR signals. One candidate is Fyn, a Src kinase coexpressed with Lck in immature and mature T cells. Here we show that T cell development is completely compromised in lck(-/-)fyn(-/-) mice. In addition, we demonstrate that expression of a gain-of-function mutant fyn(T) transgene completely restores production of immature CD4/CD8 double positive thymocytes and gammadelta T cells and improves the representation of CD4 or CD8 single positive thymocytes. These observations reveal that Fyn can subserve some Lck-like functions in T cell development.