Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress
Highlights
► The mTORC2 kinase component Protor-2 is found to interact with the mRNA destabilizing factor tristetraprolin. ► Modulation of Protor-2 levels specifically alters the stability of mRNAs known to be degraded by tristetraprolin. ► Inhibition of Protor-2 expression blocks the association of tristetraprolin with stress granules or processing bodies during stress. ► We conclude that Protor-2 regulates tristetraprolin mediated mRNA turnover and may link mTORC2 signaling to regulated mRNA stability.
Introduction
The tandem zinc-finger protein tristetraprolin (TTP; also known as Nup475, Tis11, or Zfp36) [1], [2], [3] is widely expressed and functions to regulate gene expression by binding to a conserved adenosine/uridine-rich element (ARE) within the 3ʹ untranslated region of several mRNAs [4]. TTP is known to promote mRNA deadenylation [5] and 3ʹ to 5ʹ degradation of the mRNA body [6], consistent with its ability to recruit several factors involved in these processes such as Dcp1, Dcp2 and components of the exosome [7]. The critical role for TTP in the regulation of tumor necrosis factor-α is demonstrated by the pro-inflammatory phenotype of TTP−/− mice in which chronic overexpression of TNF-α in macrophages results in severe polyarthritis and cachexia [8]. The overexpression of TNF-α in these mice is due to the stabilization of TNF-α mRNA as a result of the lack of TTP function [9]. TTP has been implicated in the posttranscriptional regulation of granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2) and cyclooxygenase 2 (COX-2) [6]. It may also regulate its own expression by binding to an ARE within the 3ʹ UTR of TTP mRNA [10]. The minimum binding site of TTP is the nonameric sequence UUAUUUAUU [11], and it is probable that additional targets of TTP containing this sequence remain to be discovered.
The p38 mitogen-activated protein kinase (MAPK) and its downstream kinase MK2 are known to activate TTP [12]. The two major sites of MK2-mediated phosphorylation of TTP identified in vitro and in vivo are serines 52 and 178 [13]. These phosphorylations result in the recruitment of 14-3-3 proteins, functional adaptors which specifically interact with particular serine- or threonine-phosphorylated proteins [14]. The recruitment of 14-3-3 proteins leads to the exclusion of TTP from stress granules (SGs) and mRNA processing bodies (P-bodies), cytoplasmic structures at which translationally stalled transcripts accumulate and mRNAs are degraded, respectively, under conditions of cellular stress [12]. The phosphorylation of TTP and its exclusion from SGs and P-bodies are associated with stabilization of ARE-containing mRNAs [1], [12].
TOR (target of rapamycin) kinase is a highly conserved, central sensor of cell growth [15]. In humans, dysregulated mTOR signaling plays a role in cancer development and progression, as well as the response to mitogens, nutrients and chemotherapeutic agents [16]. Aberrant mTOR signaling has also been implicated in tuberous sclerosis complex and lymphangiomyelomatosis [17]. TOR is found, in yeast to humans, in at least two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TORC2 [18]. The mammalian TOR complex 1 (mTORC1) contains mTOR, mLST8 and Raptor and is rapamycin sensitive. MTORC2 consists of Rictor, mSIN1, mLST8, Protor and mTOR and is rapamycin insensitive [19].
Previous studies have provided evidence that the TOR signaling cascade controls mRNA turnover in yeast [20], [21]. It has been shown that blocking TOR signaling either through nutrient limitation or rapamycin treatment causes accelerated turnover of a subset of mRNAs while others appear unaffected. More recently, several factors which regulate mRNA decay have been shown to be potential substrates of mTOR [22]. Here we demonstrate that the mTORC2 component Protor-2 interacts with TTP to promote mRNA turnover of transcripts known to degrade via TTP. We also demonstrate that Protor-2 and TTP interact in vivo and that overexpression of Protor-1 accelerates the decay of TTP-associated transcripts. Moreover, knockdown of TTP expression inhibited TTP-mediated mRNA turnover and reduced the ability of TTP to associate with SGs and P-bodies following stress induction.
Section snippets
Plasmids, cell lines and reagents
The coding region of Protor-2 was subcloned from an EST clone obtained from the German Resource Center for Genome Research (DKFZp686N03132) and cloned in frame with a C-terminal myc-epitope tag into pTRACER (Invitrogen, Carlsbad, CA). HeLa and Jurkat cell lines were obtained from ATCC. The generation of tetracycline-inducible shRNA TSCsiJurkat (T-RExJurkat, Invitrogen) cells was as described in [23]. Actinomycin D (Sigma) was dissolved in water and used at a final concentration of 10 μg/ml for
Protor-2 interacts with TTP in a yeast two-hybrid assay
To begin to identify new TTP binding proteins, we carried out a yeast two-hybrid screen using the C-terminal mRNA decay domain of human TTP (aa 174–326, C-term TTP) as bait screened against several Gal4-activation domain (Gal4AD) libraries, in addition to one constructed from cDNAs generated from mRNAs isolated from Jurkat cells (TNF-stimulated). A fusion of either the full-length, N-terminal mRNA decay domain (aa 1–100) or RNA-binding domain (aa 100–173) of the human TTP sequence to the
Discussion
Our previous data have demonstrated that TTP plays an important role in the regulation of mRNA stability in response to mTOR inhibition [24]. Here we demonstrate that TTP function is linked to TOR activity via an mTORC2 component Protor-2. Protor-2 was identified as an interactor with TTP in a yeast two-hybrid screen and the two proteins were subsequently shown to be co-immunoprecipitatable in whole cell extracts. Binding of Protor-2 to TTP was shown to require sequences within the C-terminal
Acknowledgments
We thank Drs. Michael Hall and William Rigby for providing cell lines and antibodies, Dr. Robert Nishimura for critical reading of the manuscript and Ardella Sherwood for excellent administrative assistance. This work was supported, in part, by grants from the NIH (RO1CA109312) and the US Department of Veterans Affairs.
References (45)
- et al.
Trends in Biochemical Sciences
(1995) - et al.
Immunity
(1996) - et al.
Journal of Biological Chemistry
(2004) - et al.
Journal of Biological Chemistry
(2007) - et al.
Journal of Biological Chemistry
(2004) - et al.
Seminars in Cancer Biology
(2006) - et al.
Cancer Cell
(2007) - et al.
Methods in Enzymology
(2002) - et al.
Journal of Biological Chemistry
(2007) - et al.
Methods
(2001)