Loss of function of the guanine nucleotide binding protein RhoA blocks pre-T cell differentiation and survival indicating that this GTPase is a critical signaling molecule during early thymocyte development. Previous work has shown that the Rho family GTPase Rac-1 can initiate changes in actin dynamics necessary and sufficient for pre-T cell development. The present data now show that Rac-1 actions in pre-T cells require Rho function but that RhoA cannot substitute for Rac-1 and induce the actin cytoskeletal changes necessary for pre-T cell development. Activation of Rho is thus not sufficient to induce pre-T cell differentiation or survival in the absence of the pre-T cell receptor (TCR). The failure of RhoA activation to impact on pre-TCR-mediated signaling was in marked contrast to its actions on T cell responses mediated by the mature TCR alpha/beta complex. Cells expressing active RhoA were thus hyperresponsive in the context of TCR-induced proliferation in vitro and in vivo showed augmented positive selection of thymocytes expressing defined TCR complexes. This reveals that RhoA function is not only important for pre-T cells but also plays a role in determining the fate of mature T cells.

This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN-/- T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.

Phosphatidylinositol 3'-hydroxyl kinase (PI 3-kinase) is activated by many growth factor receptors and is thought to exert its cellular functions through the elevation of phosphatidylinositol (3,4,5)-triphosphate levels in the cell. PI 3-kinase is required for growth-factor induced changes of the actin cytoskeleton which are mediated by the GTPases Rac and Rho. Recently, a role for Rac and Rho in regulating gene transcription has become evident.

Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including Fc epsilon R1 activation of mast cells. Antigenic cross-linking of the Fc epsilon R1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the Fc epsilon R1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the "classical" Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for Fc epsilon R1 induction of Elk-1. Ras function is required, but not sufficient for Fc epsilon R1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for Fc epsilon R1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.

Transcription factors of the nuclear factor of activated T cells (NFAT) family play a key role in antigen receptor-mediated responses in lymphocytes by controlling induction of a wide variety of cytokine genes. The GTPases Ras and Rac-1 have essential functions in regulation of NFAT transcriptional activity in the mast cell system, where Fcepsilon receptor type 1 (FcepsilonR1) ligation results in induction of multiple NFAT target genes. This report examines the precise biochemical basis for the Rac-1 dependency of FcepsilonR1 activation of NFAT in mast cells. We are able to place Rac-1 in two positions in the signaling network that regulates the assembly and activation of NFAT transcriptional complexes in lymphocytes. First, we show that activity of Rac-1 is required for FcepsilonR1-mediated NFATC1 dephosphorylation and nuclear import. Regulation of NFAT localization by the FcepsilonR1 is a Rac-dependent but Ras-independent process. This novel signaling role for Rac-1 is distinct from its established regulation of the actin cytoskeleton. Our data also reveal a second GTPase signaling pathway regulating NFAT transcriptional activity, in which Rac-1 mediates a Ras signal. These data illustrate that the GTPase Rac-1 should now be considered as a component of the therapeutically important pathways controlling NFATC1 subcellular localization. They also reveal that GTPases may serve multiple functions in cellular responses to antigen receptor ligation.

The tyrosine kinase p56lck regulates the differentiation and proliferative expansion of pre-T cells. However, nothing is known about other signaling molecules that operate with p56lck to mediate the pleiotropic changes that occur at this stage of thymocyte development. We used a genetic strategy to examine the requirement for the GTPase Rho in p56lck-mediated signals in the thymus. By generating mice double transgenic for a constitutively activated form of p56lck (p56lckF505) and the Rho inhibitor C3 transferase we were able to compare thymocyte development in mice expressing active p56lck on a wild-type or Rho- background. Thymocytes expressing active p56lck show enhanced proliferation of pre-T cells resulting in increased numbers of late pre-T cells, however, this dramatic effect on pre-T cell proliferation is lost when the p56lck transgene is expressed in thymocytes lacking endogenous Rho GTPase function. Expression of active p56lck also generates double positive (DP) thymocytes with low levels of CD2 antigen expression. Again, p56lck cannot prevent expression of CD2 when expressed on a Rho- background. CD4(+)CD8(+) DP cells expressing active p56lck have been shown to lack functional alpha/beta-T cell receptor (TCR) complexes due to p56lck-mediated inhibition of TCR gene Vbeta-Dbeta rearrangement. This inhibition of TCR expression by active p56lck is unimpaired in the absence of Rho function. The signaling pathways that are mediated by p56lck and control thymocyte proliferation, alpha/beta-TCR and CD2 antigen expression can thus be distinguished by their dependency on Rho function.