Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation

Abstract

Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover, it was unclear whether Tob2 phosphorylation modulates the transcriptome and whether the phosphorylation is linked to Tob2's anti-proliferative function. In this study, we found that c-Jun amino-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2-PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution profiling, transcriptome-wide mRNA turnover analyses, and cell proliferation assays, we found that the phosphomimetic mutation at S254 (S254D) enhances Tob2's association with PABP, leading to accelerated deadenylation and decay of mRNAs globally. Moreover, the Tob2-S254D mutant accelerates the decay of many transcripts coding for cell cycle related proteins and enhances anti-proliferation function. Our findings reveal a novel mechanism by which Ccr4-Not complex is recruited by Tob2 to the mRNA 3' poly(A)-PABP complex in a phosphorylation dependent manner to promote rapid deadenylation and decay across the transcriptome, eliciting transcriptome reprogramming and suppressed cell proliferation.

Keywords: Ccr4–Not complex; PABP interaction; deadenylation; mRNA turnover; phosphorylation; transcriptome programming.

FIGURE 1.

JNK activation weakens Tob2–PABP interaction through phosphorylating multiple serine and threonine residues in the intrinsically disordered region (IDR) of Tob2. (A) Co-IP western blot analysis showing that expression of constitutively active JNK1 (CA-JNK1) in U2OS cells increased phosphorylation of Tob2–V5 and diminished its interaction with PABP. In contrast, Tob2 interaction with Caf1 deadenylase was not affected by CA-JNK1 expression. Transfection with empty vector and western blot probed for actin served as negative controls. Bar graph shows the relative changes of Tob2–PABP interactions in the presence of CA-JNK1 expression using the calculated relative PABP-binding index (RPBI) values as described in Supplemental Figure S1B. The RPBI for Tob2–PABP interaction in the absence of CA-JNK1 was set as 1. All data represent the normalized mean ± SD (n = 4). (B) Co-IP western blot analysis showing that anisomycin, a JNK agonist, increased Tob2–V5 phosphorylation and weakened its interaction with PABP. JNK activation was verified by detection of p-JNK by the phospho-SAPK/JNK (Thr183/Tyr185) antibody. Actin served as a negative control. Bar graph shows the RPBI values for Tob2–PABP interactions in the cells with or without anisomycin treatment. The RPBI for Tob2–PABP interaction in the cells without anisomycin treatment was set as 1. All data represent the normalized mean ± SD (n = 3). P values were obtained from t-test. Cell extracts for IP experiments were prepared from U2OS cells transiently transfected with plasmids encoding the indicated proteins. (C) Schematic diagram of the Tob2 domain structure and phosphorylation sites. Tob2–V5 immunoprecipitates were prepared from U2OS cells without (Ctrl) or with CA-JNK1 expression for mass-spectrometry mapping of phosphorylated sites in Tob2 (Supplemental Table S1).

FIGURE 2.

Effects of different mutations in the Tob2 IDR on Tob2–PABP interaction. (A) Co-IP western blot analysis showing that phosphomimetic mutation of the serine at the fourth position of the second PAM2 motif of Tob2 (S254D) enhances Tob2 interaction with PABP but has little effect on the Tob2–Caf1 interaction. Transfection with empty vector and western blot probed for actin served as negative controls. Bar graph shows the RPBI values for the relative PABP pulled down. The RPBI for wild type (WT) Tob2–PABP interaction was set as 1. All data represent the normalized mean ± SD (n = 3). (B) Co-IP western blot analysis showing that a Tob2 mutant with phosphomimetic changes of 11 ser/thr sites in the IDR (PM11) weaken the interaction between Tob2 and PABP. Transfection with empty vector and western blot probed for GAPDH served as negative controls. Bar graph shows the RPBI value for the relative PABP pulled down. The RPBI for WT Tob2–PABP interaction was set as 1. All data represent the normalized mean ± SD (n = 3). (***) P < 0.001 and (****) P < 0.0001 (t-test). (C,D) Co-IP western blotting results showing that substitution of aspartate for the serine at the fourth position of the second PAM2 motif in Tob1 (S268D) greatly enhances Tob1 interaction with PABP, whereas a similar phosphomimetic substitution in the sole PAM2 motif of PAIP1 (S129D) has little effect. Transfection with empty vector and western blot probed for actin (C) or Caf1 (D) served as negative controls. Bar graphs show the RPBI values for the relative PABP pulled down normalized to Tob1 or PAIP1. The RPBI for WT Tob2–PABP interaction was set as 1. All data represent the normalized mean ± SD (n = 2). P values were obtained from t-tests. n.s., P > 0.05, not significant in t-test. (E,F) Co-IP western blotting results showing that the two PAM2 motifs in Tob2 cooperate to expand the range of PABP-interaction strengths. Mutation of the highly conserved phenylalanine in the first (F140A) or second PAM2 motif (F260A) of Tob2 WT (E) or S254D mutant (F) greatly reduces their interactions with PABP. Double mutation (F140A/F260A; FF) of Tob2 WT (E) or Tob2–S254D (F) knocked out Tob2's ability to interact with PABP. The interaction of Tob2 with Caf1 was not affected. A schematic diagram of the arrangement of Tob2 domains and the F140A and F260A mutations is shown below panels E and F. Transfection with empty vector served as a negative control. Bar graphs show the RPBI values for the relative PABP pulled down. The RPBI for WT Tob2–PABP interaction was set as 1. All data represent the normalized mean ± SD (n = 2) except that Tob2–S254D (FF) mutant in panel F was tested only once. (**) P < 0.01 and (***) P < 0.001 (t-test). Cell extracts for IP experiments were prepared from U2OS cells transiently transfected with plasmids encoding the indicated proteins.

FIGURE 3.

Effects of JNK-induced phosphorylation and the corresponding phosphomimetic mutations on the interaction of Tob2 WT or S254D mutant with PABP. (A) Co-IP western blotting results showing that simultaneous phosphomimetic mutation at 11 ser/thr sites in the IDR to mimic JNK-induced hyper-phosphorylation of the Tob2 WT (PM11) or the Tob2–S254D mutant (PM12) weakened Tob2 interactions with PABP but not with Caf1. (B) Co-IP western blotting results showing that ectopic expression of the constitutively active JNK1 isoform (CA-JNK1) increased phosphorylation of both Tob2 WT and S254D mutant and diminished their interactions with PABP. The Tob2 interactions with Caf1 were not affected. Transfection with empty vector and western blot probed for actin served as negative controls. Bar graphs show the RPBI values for the relative PABP pulled. The RPBI for WT Tob2–PABP interaction was set as 1. All data represent the normalized mean ± SD (n = 2). (**) P < 0.01, (***) P < 0.001 and n.s. (P > 0.05; not statistically significant by t-test). Cell extracts for IP experiments were prepared from U2OS cells transiently transfected with plasmids encoding the indicated proteins.

FIGURE 4.

Phosphomimetic mutation at S254 of Tob2 further enhances Tob2's ability to promote mRNA deadenylation. (A) Northern blot and time-course experiment results showing the accelerating effects of ectopic expression of Tob2 WT or S254D mutant protein on deadenylation of β-globin mRNA. Poly(A)^– RNA samples (A⁻) were prepared with oligo(dT) and RNase H treatment. The autorads of the gels were scanned for densitometric analysis of each band using ImageJ software. RNA half-lives (t_1/2) in hours (h) were determined by least-square analysis of semilogarithmic plots of normalized mRNA concentration as a function of time. (B) Comparisons of the β-globin poly(A) shortening profiles in the presence of Tob2 WT or S254D mutant with the profile in control (Ctrl) cells. The autorads of the gels in panel A were scanned for densitometric analysis of each lane using ImageJ software. Western blot showing levels of ectopically expressed Tob2 WT and S254D mutant proteins. GAPDH served as a loading control. (C) (Upper) Denaturing gel showing changes in poly(A) size distribution of the entire mRNA population in control cells, cells ectopically expressing Tob2 protein, or cells ectopically expressing Tob2's mutant derivatives as indicated. Lane M: size markers as labeled to the right of the gel in nucleotide (nt). (Lower) Western blot showing levels of ectopically expressed Tob2 protein and its mutant derivatives. Caf1 served as a loading control. (D) The poly(A) size distribution profiles of the gel in panel C were obtained by densitometric analysis of each lane using ImageJ software. Note that a typical poly(A) size distribution profile (Ctrl) exhibits two peaks at ∼150 nt and ∼50 nt as a result of the biphasic deadenylation mediated by two different deadenylase complexes (i.e., Pan2–Pan3 and Ccr4–Not complexes), respectively. Total cytoplasmic RNA samples and cell extracts were prepared from U2OS cells transiently transfected with plasmids encoding the indicated proteins.

FIGURE 5.

Tob2–S254D elicits profound destabilization of mRNA across the transcriptome. (A) Outline of the workflow for bromouridine (BrU) metabolic labeling and transcriptome-based RNA-seq analyses of mRNA decay rates. (B) Cumulative fraction plots showing the distribution of mRNA half-lives of the qualified transcripts exhibiting strong first order decay kinetics with (blue line) and without (red line) induction of empty vector, WT Tob2 or S254D expression (Supplemental Table S4). The P values were calculated by t-test. (C) Western blot analysis showing induced WT Tob2 or Tob2–S254D and corresponding endogenous Tob2 proteins, with endogenous Caf1 serving as a loading control. (D) Combined scatter and frequency plot analysis of the half-lives of 3490 transcripts exhibiting strong first order decay kinetics with (y-axis) and without (x-axis) induction of Tob2–S254D expression. Red: most dense; dark blue: least dense. Transcripts with unchanged stability fall on or near the diagonal, dashed red line. Cell extracts and total cytoplasmic RNA samples were prepared from double-stable U2OS cells whose expression of V5-tagged WT Tob2, Tob2–S254D, or vector only is controlled by the Tet-off promoter (see Materials and Methods for details).

FIGURE 6.

Effects of expression of Tob2 WT or Tob2–S254D mutant on cell proliferation. (A) Cell growth assay of control U2OS cells and cells expressing Tob2 WT or the Tob2–S254D mutant. Results shown represent three biological repeats, each consisting of eight technical repeats. All data in bar graph represent the normalized mean ± SD (n = 3). (*) P < 0.05 and n.s. (not statistically significant; P > 0.05) by t-test. (B) Clonogenic assay of colony forming abilities of control U2OS cells and cells induced to express Tob2 WT or the Tob2–S254D mutant. (Left) Colonies visualized by crystal violet staining after 12 d of growth under uninduced (+tetracycline) or induced (−tetracycline) conditions. (Right) Phase-contrast images of colonies taken prior to crystal violet staining. Red bar: 250 µm. (C) Western blot analysis of Tob2 protein expression, with GAPDH as a loading control. Quantitation of the western blot results from three repeats indicates that the slight difference of WT Tob2 versus S254D mutant protein levels normalized to GAPDH is not statistically significant (n = 3; P = 0.50; t-test).

FIGURE 7.

A hypothetical model for phosphorylation-dependent regulation of Tob2 function. Refer to the Discussion for details. (A) Three different patterns of Tob2 phosphorylation that weaken or strengthen the Tob2–PABP interaction. (B) Tob2 phosphorylation at S254 proposed to favor alignment of the two PAM2 motifs, strengthening interactions with PABP and optimally positioning the Ccr4–Not complex and the 3′ poly(A)–PABP complex to facilitate poly(A) removal. For simplicity, some known phosphorylation sites in the IDR are omitted from the cartoons.