Activation of extracellular-signal-regulated protein kinase (Erk) is central to growth-factor-receptor-mediated signaling including that originating from the T cell antigen receptor. It integrates cytoplasmic signals to effect changes in transcription associated with differentiation, proliferation, and survival. In this report, we present an analysis of mice with targeted deletions in Erk1 and Erk2 to assess the relationship between Erk activity and cell-cycle progression, thymocyte development, and lineage commitment. These studies show that Erk is selectively retained during beta selection-driven proliferation, and yet Erk1/2 are not required to complete differentiation to CD4+CD8+ preselection stage of development. Erk activity is essential for the process of positive selection, and it differentially affects CD4 and CD8 T cell maturation; yet, diminished expression itself is not sufficient to alter lineage commitment.

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.One fundamental role of the yeast vacuole is in the recycling of biological macromolecules. The vacuole, like the lysosome in animal cells, is the primary site of degradation. Our understanding of the hydrolytic vacuolar enzymes that serve in protein turnover is well advanced. Progress has also been made in elucidating mechanisms that deliver the substrates of these vacuolar hydrolases. While research has focused on the biosynthesis and function of the vacuolar proteases, little is known about how lipids are recycled in this organelle, and a lipase that functions in membrane recycling has not been identified.Nearly all vacuolar/lysosomal delivery pathways involve packaging of cargo within membrane-enclosed transport compartments. Because the vacuole/lysosome serves as the final destination for these numerous vesicle-mediated transport pathways, the issue of how membranes reaching the vacuole are recycled is an important one. Macroautophagy is the major degradative process in eukaryotes and is essential during starvation conditions (1). In yeast, autophagy overlaps with a biosynthetic process, the Cvt pathway, that delivers the hydrolase aminopeptidase I (API 1 ; Ref.2) from its site of synthesis in the cytoplasm to the vacuolar lumen. Cvt and autophagy employ many of the same molecular components and are mechanistically related (3-6). Both pathways involve the formation of double-membrane cytosolic vesicles, sequestering either precursor aminopeptidase I (prAPI) specifically, or in the case of autophagy, also enveloping bulk cytosol in a nonselective manner. Fusion of these vesicles with the vacuole results in the release of single-membrane subvacuolar vesicles within the lumen. These pathways require a mechanism for specific lysis of the internalized vesicles, so that vesicle cargo can be released into the vacuole lumen, and further require a mechanism for degradation of vesicle lipids.To understand the molecular basis of these import and degradation pathways, we carried out a g...

The magnitude of T cell responses is determined by proliferation and survival decisions made by the responding cells. We now demonstrate that the Erk MAPK pathway plays a critical role in these cell fate decisions within CD8 T cells. While Erk1 is dispensable for all aspects of CD8 T cell activation, Erk2 is required for the proliferation of CD8 T cells activated in the absence of costimulation. Surprisingly, Erk2 is not required for proliferation following the addition of a costimulatory signal in vitro, or upon viral infection in vivo, but regulates the size of the responding population by enhancing cell survival. An important component of this Erk2-derived signal is the transcriptional regulation of Bcl-2 family members Bcl-xL and Bim, and impaired Erk2-deficient CD8 T cell survival can be rescued by genetic ablation of Bim. These studies ascribe multifaceted functions specific to Erk2 in CD8 T cell activation, proliferation, and survival.

Cancer chemotherapy-related symptoms such as fatigue, malaise, loss of interest in social activities, difficulty concentrating, and changes in sleep patterns can lead to treatment delays, dose reductions, or termination and have a profound effect on the physical, psychosocial, and economic aspects of quality of life. Clinicians have long suspected that these symptoms are similar to those associated with "sickness behavior," which is triggered by the production of the inflammatory cytokines IL-1beta, TNF-alpha, and IL-6 by macrophages and other cells of the innate immune system in response to immune challenge. The p38 mitogen-activated protein kinase (p38 MAPK) plays a central role in the production of these cytokines and consequently the induction of sickness behavior. Several cancer chemotherapy drugs have been shown to activate p38 MAPK, but whether these drugs can also induce the production of inflammatory cytokines to cause sickness behavior is unknown. The aim of this study was to determine whether the cancer chemotherapy drug etoposide (VP-16), which is known to activate p38 MAPK, could induce inflammatory cytokine production by murine macrophages and sickness-like behaviors when injected into mice. VP-16 activated p38 MAPK and induced IL-6 production in murine macrophages in a p38 MAPK- dependent manner. VP-16 administration rapidly increased serum levels of IL-6 in healthy mice and induced sickness-like behaviors as evidenced by a decrease in food intake, body weight, hemoglobin level, and voluntary wheel-running activity. These findings support the idea that the induction of IL-1beta, TNF-alpha, and IL-6 by cancer chemotherapy drugs underlies the fatigue and associated symptoms experienced by people undergoing cancer chemotherapy.

Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.