Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.

The transcription factor, Nuclear Factor of Activated T cells (NFAT) is a major target for p21ras and calcium signalling pathways in the IL‐2 gene and is induced by p21ras signals acting in synergy with calcium/calcineurin signals. One p21ras effector pathway involves the MAP kinase ERK‐2, and we have examined its role in NFAT regulation. Expression of dominant negative MAPKK‐1 prevents NFAT induction. Constitutively active MAPKK‐1 fully activates ERK‐2 and the transcription factor Elk‐1, but does not substitute for activated p21ras and synergize with calcium/calcineurin signals to induce NFAT. Expression of dominant negative N17Rac also prevents TCR and p21ras activation of NFAT, but without interfering with the ERK‐2 pathway. The transcriptional activity of the NFAT binding site is mediated by a complex comprising a member of the NFAT group and AP‐1 family proteins. The induction of AP‐1 by p21ras also requires Rac‐1 function. Activated Rac‐1 could mimic activated p21ras to induce AP‐1 but not to induce NFAT. Moreover, the combination of activated MAPKK‐1 and Rac‐1 could not substitute for activated p21ras and synergize with calcium signals to induce NFAT. Thus, p21ras regulation of NFAT in T cells requires the activity of multiple effector pathways including those regulated by MAPKK‐1/ERK‐2 and Rac‐1.

The present study employs a genetic approach to explore the role of Rho GTPases in murine thymic development. Inactivation of Rho function in the thymus was achieved by thymic targeting of a transgene encoding C3 transferase from Clostridium botulinum which selectively ADP‐ribosylates Rho within its effector domain and thereby abolishes its biological function. Thymi lacking functional Rho isolated from C3 transgenic mice were strikingly smaller and showed a marked (90%) decrease in cellularity compared with their normal litter mates. We also observed a similar decrease in levels of peripheral T cells in C3 transgenic mice. Analysis of the maturation status of thymocytes indicated that differentiation of progenitor cells to mature T cells can occur in the absence of Rho function, and both positive and negative selection of T cells appear to be intact. However, transgenic mice that lack Rho function in the thymus show maturational, proliferative and cell survival defects during T‐cell development that severely impair the generation of normal numbers of thymocytes and mature peripheral T cells. The present study thus identifies a role for Rho‐dependent signalling pathways in thymocyte development. The data show that the function of Rho GTPases is critical for the proliferative expansion of thymocytes. This defines a selective role for the GTPase Rho in early thymic development as a critical integrator of proliferation and cell survival signals.

Resting T lymphocytes that have recognized antigen bound to a major histocompatibility complex molecule with the T-cell receptor require ciniulatory through accessory receptors, includg CD2, CD4, CD8, and CD28, for their clonal growth and expression of their functional repertoires. Absence of costimulation, in contrast, can induce clonal anergy in vitro and selective tolerance in vivo. Here we have defined a potential intracellular messenger for T-cell activation which-is strictly regulated by costimulatory signals mediated through accessory receptors: ppl9/cofilin, a small actinbinding protein, undergoes dephosphorylation and subsequent translocation from the cytosol into the nucleus. In untransformed T cells this process correlates with functional responses essential for the induction of T-cell proliferation (i.e., production of interleukin 2). Moreover, spontaneous dephosphorylation as well as nuclear translocation ofppl9/cofllin occur in the autonomously proliferating T-lymphoma cell line Jurkat.Engagement of the T-cell receptor (TCR)/CD3 complex by an antigen/major histocompatibility complex (MHC) molecule produces a "first signal" for T-cell activation which results in expression of the interleukin 2 (IL-2) receptor (1). However, whether encounter of antigen/MHC leads to clonal T-cell expansion or, alternatively, clonal anergy (unresponsiveness) depends on the availability of a costimulatory signal or signals. Costimulatory signals can originate from ligand attachment to accessory receptors like CD2, CD4, CD8, or CD28. They are required for the induction of responsiveness to monokines and production of the T-cell growth factor IL-2 (2-10). Intracellular messengers of costimulatory functions-i.e., components of signaling pathways selectively engaged through accessory receptors-have not yet been defined.We have recently observed a cytoplasmic phosphoprotein (ppl9) in human T lymphocytes whose phosphoserine residues are dephosphorylated in response to accessory receptor triggering (i.e., activation by CD2 or CD3 x CD4 or CD3 x CD8 crosslinking), but not after TCR/CD3 stimulation alone (2, 11). Dephosphorylation of ppl9 correlates with the induction of interleukin 6 responsiveness and secretion of IL-2. In contrast, interferon fy (IFN-y) production is inducible to a similar degree through TCR/CD3 triggering alone (2). The latter parameter was used to ensure that-despite their differential activities regarding the observed dephosphorylation event-stimulating monoclonal antibodies directed at TCR/CD3 and the accessory receptors CD2, CD4, or CD8, respectively, exhibited comparable intrinsic triggering capacities. The human T-cell clone (TCR a/.B/CD3+; CD2+; CD4+; CD8-) was generated as described (12). The human T-lymphoma line Jurkat was maintained in RPMI 1640 medium containing 10%o fetal calf serum. Ascites fluids containing monoclonal antibodies against different epitopes of the human CD2 molecule [T111A (3T48B5), T112 (1OLD4C1), and T113 (lmono2A6)(4)] were kindly provided by S. F. Schlossman (Dana-Fa...