T lymphocytes expressing the T cell receptor (TCR)-γδ recognize unknown antigens on tumor cells. Here we identify metabolites of the mevalonate pathway as the tumor ligands that activate TCR-γδ cells. In tumor cells, blockade of hydroxy-methylglutaryl-CoA reductase (HMGR), the rate limiting enzyme of the mevalonate pathway, prevents both accumulation of mevalonate metabolites and recognition by TCR-γδ cells. When metabolite accumulation is induced by overexpressing HMGR or by treatment with nitrogen-containing bisphosphonate drugs, tumor cells derived from many tissues acquire the capacity to stimulate the same TCR-γδ population. Accumulation of mevalonate metabolites in tumor cells is a powerful danger signal that activates the immune response and may represent a novel target of tumor immunotherapy.

In yeast TOR and PKA pathways both control cell growth but how TORC1 and PKA signaling are linked is unknown. Here we show that TORC1 inhibition prevents the phosphorylation of some but not all PKA targets. We further demonstrate that TORC1 controls PKA by inhibiting the phosphorylation of the PKA regulatory subunit BCY1 by the MAP kinase MPK1.

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TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFα and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

We have generated a monoclonal antibody (mAb), 9EG7, against mouse endothelial cells that blocks adhesion of lymphocytes to endothelial cells. Sequencing offour tryptic peptides ofthe purified antigen revealed its identity with the integrin chain (3. The only Pu integrin that is known to mediate cell-cell adhesion is a4fi . This is not the integrin that is functionally dermed by the mAb 9EG7 on endothelial cells. First Antibodies. The following mAbs against mouse integrin chains were used: PS/2 (24) (anti-a4), R1-2 (16) (anti-a4), GoH3 (25) (anti-a6), and EA-1 (26, 27) (anti-a6). Other mAbs were K20 (28) (anti-human integrin chain (31) and (anti-mouse VCAM-1). Rabbit antibodies against a peptide corresponding to the C-terminal domain of the chicken integrin chain (31 were obtained from Richard 0. Hynes (MIT).The mAb 9EG7 (IgG2a) was produced by immunizing rats with TME cells in phosphate-buffered saline (PBS), using Alu-Gel-S (Serva) as adjuvant. Hybridoma production was done essentially as described (30) except that the mouse myeloma Sp2/0 was used for the fusion. Hybridoma supernatants were screened in cell-surface ELISA assays on TME Abbreviations: CAM, cell adhesion molecule; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate.lTo whom reprint requests should be addressed. 9051