Protor-2 interacts with tristetraprolin to regulate mRNA stability during stress

Abstract

The A/U-rich RNA-binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of Rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP-regulated mRNA stability to mTORC2 activity.

Figure 1

Y2H mapping of interacting regions of Protor-2 and TTP. The indicated deletion mutants of Gal4DBD-TTP or Gal4AD-Protor-2 were cotransfected into AH109 cells and plated onto selective media in the absence of histidine to determine whether an interaction between the proteins was detectable via activation of the HIS3 reporter (+++, positive growth; ++, moderate growth; −, no growth). Colonies which grew were assayed for β-gal activity as described (XX).

Figure 2

Protor-2 interacts with TTP in vivo. TTP, Protor-2 or Rictor was immunoprecipitated from Jurkat or TSCsiJurkat cells as indicated. Western blots were subsequently performed on the immunoprecipitates for the indicated proteins. Lane 1, beads without antibody; lane 2, immunoprecipitation using irrelevant antibody (actin Ab), lane 3, input uninduced Jurkat cell lysate, lane 4, immunoprecipitation antibody, lane 5, TSCsiJurkat cell lysate with immunoprecipitation antibody.

Figure 3

Overexpression of Protor-2 promotes TTP-associated mRNA turnover. A. Expression of myc-tagged Protor-2 in Jurkat cells. Lane 1, Jurkat_Puro control transfectants, Lane 2, Jurkat_Protor2 overexpression clone. Western blot was probed with a-myc epotope antibodies (4E11) and actin. B. Relative mRNA turnover and half-lives (t_1/2) of the indicated transcripts from Jurkat_Puro (shaded circles) or Jurkat_Protor2 (shaded squares) as determined by ActD-mRNA half-life experiments followed by real time qRT-PCR of the indicated transcripts. C. Relative mRNA turnover of the indicated mRNAs in Jurkat_Zeo (open circles) versus TSCsiJurkat (shaded triangles) cells following tet-induced suppression of TSC1/TSC2.

Figure 4

Effect of Protor-2 knockdown on TPP-associated mRNAs during stress-induced destabilization. A. Jurkat cells were transfected with control, non-targeting scrambled sequence or Protor-2-targeting siRNAs and immunoblotted for the indicated proteins. B. Relative mRNA turnover and half-lives (t_1/2) of Jurkat cells treated with FCCP (10 µM) and transfected with control, scrambled sequence or Protor-2-targeting siRNAs as indicated.

Figure 5

Inhibition of Protor-2 expression reduces TTP association with stress granules and P-bodies. HeLa cells were treated with control, non-targeting or Protor-2-targeting siRNA and treated with FCCP (1 µM) to induce SG and P-bodies formation. Subcellular localization of TTP and stress granule (TIA-1) and P-body (Dcp1) markers (right panels, red, as indicated) were determined by dual label immunofluorescence using CARP-3, an affinity-purified antibody reactive with TTP (left panels, green). Arrows point out SGs and P-bodies. Bar size is 10 µm.