Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes

Abstract

Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2-dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4 ؉ T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer The underlying principle of cancer immunotherapy is to eliminate malignant cells by tuning the immune system (1-2). This revolutionary way of fighting tumors was originated three decades ago when a patient suffering from metastatic melanoma was treated with the T-cell growth promoting factor interleukin-2 (IL-2) 1 (3). The success of IL-2 administration in fighting metastatic melanoma demonstrated for the first time that solely potentiating the activation of T lymphocytes could abrogate certain human cancers (4). Current immunotherapy approaches include the use of autologous gene-engineered T cells that, once expanded ex vivo with IL-2, are re-infused back into patients by the so-called adoptive cell transfer therapy (ACT) (5-7). Despite the promising results of this approach, a safe and long-lasting expansion of transferred T cells remains a major challenge because of the undesirable side effects derived from the use of IL-2. Continued exposure to high doses of IL-2 results in increased susceptibility of T cells to apoptosis (8). Moreover, IL-2 is also a critical component for regulatory T-cell (T reg ) development and function (9 -10), and as such it functions as a negative regulator of the immune response (11). Consequently, although IL-2 constitutes a key component of current immunotherapies, a great deal of effort is being devoted to the development of novel strategies that would boost the T-cell immune response more safely. In this regard, it has been shown that IL-2-related toxicity can be partially minimized by the use of gene-engineered T cells ex...

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics

“…For instance, in our last study focused on deciphering the nuclear phosphoproteome of Tlymphocytes we detected 8,521 unique phosphosites of which more than 10% were not reported in the Phosphosite database and hence were considered as novel [24]. Comparing our phosphosite-panel with the PPSA commercialized by Cell Signaling, we concluded that at present less than 1% of our phosphosites could potentially be detected and therefore verified by the PPSAs available to date.…”

“…Genetic approaches such as site-directed mutagenesis have greatly expanded our knowledge about biological role of the site specific phosphorylations occurring in proteins [22][23][24]. The increasing number of commercially available phosphosite-specific antibodies also allows detecting relatively easily whether a protein is phosphorylated or not on a certain residue that has been previously described.…”

Nola eragiten du 2-Interleukinak T-zelulen nukleoko proteinen fosforilazio mailan?

“…Horrenbestez, lan-fluxu honen abantailez baliatu gara IL-2k T-zelulen nukleoko proteinen fosforilazio maila nola erregulatzen duen zehazteko [37].…”

“…Guztira fosforilatutako 8521 aminoazido identifikatu eta kuantifikatu ziren egindako esperimentu beraren 3 saiotan [37]. Oro har, fosforilatuta…”

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells

“…In these studies, Osinalde and co-workers utilized SILAC labeling and high resolution MS combined with phosphotyrosine immunoprecipitation [91][92][93] and TiO2 enrichment of the phosphopeptides [94,95] to demonstrate that the signaling pathways activated by IL-2 and IL-15 are very similar [94,95]. In addition, they combined antibody-and TiO2-based enrichment with SILAC labeling before MS analysis to show that serine/threonine phosphorylation of the scaffold protein Gab2 is an important step in regulating IL-2 signaling [96] Quantitative phosphoproteomics has also been used to characterize CD8+ CTLs in detail [97,98].…”