No abstract

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysomebound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [35 S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.Rapamycin is a macrolide antibiotic originally isolated from Streptomyces hygroscopicus (1). It is a potent immunosuppressant with therapeutic applications in the prevention of organ allograft rejection and in the treatment of autoimmune disease (2-6). The importance of rapamycin as an immunosuppressant drug has focused attention on its mechanism of action. Rapamycin has a similar biochemical structure to cyclosporin A and FK506. However, unlike cyclosporin A and FK506, rapamycin is not a calcineurin inhibitor (7). The primary mode of immunosuppressive action of rapamycin is an antiproliferative action reflecting the ability of the drug to disrupt signaling by T cell growth-promoting lymphokines such as IL-2 1 and IL-4 (8). The growth-inhibitory effects of rapamycin are not limited to T cells, since this drug inhibits the proliferation of many mammalian cell types as well as that of yeast cells (9).Rapamycin blocks progression of the cell cycle at the G 1 phase by binding to FKBP12 (FK506-binding protein) (10, 11). The rapamycin-FKBP12 complex inhibits mTOR (mammalian target of rapamycin), also referred to as FRAP (FKBP-rapamycin-associated protein) (9). Targets of mTOR include 4E-BP1 and the 40 S ribosomal protein S6 kinase, p70 s6k (12-16). Rapamycin-induced inhibition of p70 s6k activity and subsequent dephosphorylation of the ribosomal S6 protein lead to a selective translational repression of mRNA containing a polypyrimidine-rich tract (TOP) motif at their 5Ј terminus (17). 4E-BP1 is a small heat-and...

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGKα, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGKα, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGKα to the membrane fraction. At early time points, DGKα translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGKα activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGKα mutants. We show that membrane localization of DGKα is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGKα, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.