IL-2– and IL-15–induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

Abstract

Introduction mTOR (mammalian target of rapamycin) is a ubiquitously expressed serine/threonine kinase that affects a number of key cell functions, including protein synthesis and proliferation. 1,2 mTOR associates with either protein called raptor or another named rictor to form the mTORC1 and mTORC2 complexes, respectively. The signaling pathways activated by mTORC1 have thus far been much better characterized. 1-3 Accordingly, mTORC1 acts by activating p70S6 kinase 1 (p70S6K1) and inhibiting the 4E binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates an S6 protein of the 40S ribosomal subunit (S6rp) at several sites, including serines 235 and 236. The exact mechanisms of mTOR activation are still under investigation, but at least 2 separate, distinct signals are required. Whereas the first is provided by the cell membrane receptors for growth factors, such as insulin and insulin-like growth factor (IGF), the second is generated by nutrients. [4][5][6][7] The IGF and other receptors activate cell-signaling pathways PI3K/Akt 8-10 and ERK/ MEK. 11-13 Both these signaling pathways have been implicated in mTORC1 activation through an indirect mechanism by suppressing the activity of the tuberous sclerosis complex proteins TSC1 and TSC2 which, in turn, inhibit activity of mTORC1 through inactivating the G protein Rheb. mTORC1 can be functionally inactivated by inhibitors from the rapamycin family. Rather than occupying the enzymatic kinase/ATP-binding domain, rapamycin-type compounds block the binding of mTORC1 to the FKBP12 protein and, consequently, inhibit activity of the complex. Rapamycin and its derivatives are highly potent and specific for mTORC1. They are currently used clinically as immunosuppressive drugs and are evaluated as therapeutic agents in various types of cancer. 1,2 From the several cytokines such as IL-2, IL-7, IL-15, and IL-21 that signal through the receptors that share the common ␥ chain (␥c), IL-2 is by far the best characterized. It signals through the receptor that, in addition to the ␥c, contains the second signaling chain ␤ and, in the case of high-affinity receptor, also the IL-2-specific, signal nontransducing ␣ chain. In normal immune cells, IL-2 has been shown to activate Jak/STAT signaling, PI3K/Akt and MEK/ERK signaling pathways. 14 In addition to the ␥c, the receptor for IL-7 contains a second IL-7-specific signaltransducing ␣ chain. IL-7 has been implicated in promoting maturation and survival of T lymphocytes. [15][16][17][18] In turn, IL-15 shares with IL-2 both receptor signaling chains, ␥ and ␤, and, similar to IL-2, also has the IL-15-specific, nontransducing ␣ chain. Consequently, IL-2 and IL-15 share a number of properties, including stimulation of the T-, natural killer (NK)-, and B-cell proliferation and functional maturation, but certain features unique to each of them have also been described. 14,19 IL-21 also displays a spectrum of effects on the immune cells, 20 with its ability to increase cytotoxicity of both NK 21 and CD8 ϩ T 22 cells being the best define...

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

“…We used in this analysis the phospho-specific antibodies against S6rp (Ser235/236) and p-4E-BP1 (Ser65), which we have validated extensively in our recent studies. [23][24][25] Whereas these antibodies have proven highly suitable for immunohistochemistry, an antibody against phosphorylated p70S6K (Thr389) did not, due to its cross-reactivity with p-p90S6K, which is not phosphorylated by mTORC1. 25 The representative results of the current evaluation of the mTORC1 signaling pathway activation in ARL are depicted in Figures 1 to 3.…”

“…[23][24][25] In brief, 5-m sections from formalin-fixed, paraffin-embedded tissue specimens were deparaffinized in xylene and rehydrated in graded alcohol. After the deparaffinization, heat induced antigen retrieval was performed by boiling the slides in 10 mol/L citrate buffer for 20 minutes.…”

“…22,23 The LY18 and Val lines were derived from the DLBCL of germinal center origin. The Raji, Daudi, and Ramos cell lines were obtained from BL.…”

“…[22][23][24] In brief, the cells were washed briefly in PBS, centrifuged and lysed in an radioimmunoprecipitation assay buffer supplemented with 0.5 mmol/L phenylmethylsulfonyl fluoride, phosphatase inhibitor cocktails I and II from Sigma and protease inhibitor cocktail from Roche (Nutley, NJ), according to the manufacturer's specifications. For normalization of the gel loading, the protein extracts were assayed with the Lowry method (Bio-Rad Dc protein assay).…”

“…21 Although inhibitors of either PI3K/Akt or MEK/ERK signaling pathways did fully inhibit mTORC1 activation in transformed B lymphocytes, at least when applied alone, 22 these pathways have been found to contribute to mTORC1 stimulation in two types of T-cell lymphoma. 23,24 In this study, we identified the activation of the mTORC1 pathway in all ARL cases examined, regardless of their specific histological classification and immunophenotype. mTORC1 was also activated in the hyperplastic follicles of the HIV-associated lymphadenopathy.…”