The Foxo transcription factors (Foxo1, Foxo3, Foxo4) modulate cell fate decisions in diverse systems. Here we show that Foxo1-dependent gene expression was critical at multiple stages of B cell differentiation. Early deletion of Foxo1 caused a severe block at the pro-B cell stage, due to a failure to express interleukin 7 receptor α (IL-7Rα). Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to a reduction in Rag1 and Rag2 expression. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to reduced L-selectin expression, and failed class switch recombination due to impaired Aicda upregulation. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.

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The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes with a recombinant membrane-permeable YopH resulted in severe reduction in intracellular tyrosine phosphorylation and inhibition of T cell activation. The primary signal transducer for the T cell antigen receptor, the Lck tyrosine kinase, was specifically precipitated by a substratetrapping YopH mutant, and Lck was dephosphorylated at its positive regulatory site, Tyr-394, in cells containing active YopH. By turning off Lck, YopH blocks T cell antigen receptor signaling at its very first step, effectively preventing the development of a protective immune response against this lethal bacterium.

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar K i values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopHinduced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.

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The stress activated protein kinase pathway culminates in c‐Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP‐1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3‐deficient mice and quantified nerve growth factor (NGF) deprivation‐induced neuronal death, oxidative stress, c‐Jun phosphorylation, and c‐jun induction. Here, we report that oxidative stress in neurons from JNK3‐deficient mice is normal after NGF deprivation. In contrast, NGF‐deprivation‐induced increases in the levels of phosphorylated c‐Jun, c‐jun, and apoptosis are each inhibited in JNK3‐deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c‐Jun and apoptosis in a classic model of cell‐autonomous programmed neuron death.

Caveolae are cholesterol-rich, membrane microdomains that appear critical to signaling between extracellular and intracellular macromolecules as well as cholesterol homeostasis. Caveolae formation is modulated by caveolin, a protein family that is the proteinaceous hallmark of caveolae. Very little is known regarding the events that modulate caveolin expression and regulation in neurons. To detect caveolin expression in neurons, primary rat hippocampal neurons were harvested at embryonic day 18, maintained for 7 days in vitro, and then analyzed for caveolin immunofluorescence. Caveolin-1 immunoreactivity was detected in cells that were identified as neurons by morphology and concurrent microtubule-associated protein (MAP2) staining. Changes in caveolin-1 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of RNA isolated from hippocampal neurons treated with glutamate receptor agonists. Glutamate induced a concentration-dependent increase in caveolin-1 mRNA. The largest increases in caveolin-1 mRNA were detected after 6 hours of treatment. Kainate and AMPA both mimicked glutamate effects on caveolin-1 mRNA expression. Western blot analyses revealed that caveolin was induced at the protein level as well. Taken together, these data suggest that glutamate can regulate caveolin expression through kainate and AMPA ionotropic glutamate receptors.