PTEN arginine methylation by PRMT6 suppresses PI3K-AKT signaling and modulates pre-mRNA splicing

Abstract

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K-AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K-AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.

Keywords: PI3K–AKT cascade; PRMT6; PTEN; arginine methylation; pre-mRNA splicing.

Fig. 1.

Dimethylation of PTEN R159 is required for repression of PI3K–AKT signaling. (A) Raw data for missense mutations of the PTEN gene in sporadic cancer samples were derived from the COSMIC database. There were a total of 2,271 clinical samples with missense mutations of the PTEN gene (updated on August 22, 2018), and the mutation rate of each type of amino acid was analyzed (A, Left). (A, Right) The analysis was also normalized to the amino acid constitution of PTEN protein. (B) Evolutionary conservation of the PTEN R159 residue was analyzed with ClustalX. Sequence data of PTEN from different species were derived from the UniProt database. (C) S-tagged PTEN proteins were precipitated from cell lysates with S-protein beads and subjected to PTEN enzyme reactions (PIP₃ substrate). OD_620nm reflects the quantity of PO₄ released in the reaction. Data were analyzed by using the paired two-tailed Student’s t test. Error bars indicate SEM (n = 3). ns, no significant difference; ***P < 0.001. (D–G) pAKT (S473) levels in PTEN^−/− U2OS (D), PTEN-null U-87 MG (E), H4 (F), and PC-3 (G) cells expressing S-tag control, wild-type PTEN (PTEN-WT), PTEN R159K, PTEN C124S, or PTEN G129E were analyzed by using immunoblotting. Levels of pAKT (S473) and total AKT expression were analyzed. GAPDH served as a loading control.

Fig. 2.

PRMT6 is involved in repression of the PI3K–AKT cascades. (A) Exogenous methyltransferases PRMT1–9 were individually introduced into PTEN^−/− U2OS cells reexpressing wild-type PTEN (Left) or control S-tag (Right). pAKT (S473) and total AKT levels were analyzed. GAPDH served as a loading control. pAKT (S473) quantification relative to total AKT is shown below the blots. Data were analyzed with the unpaired two-tailed Student’s t test. Error bars indicate SEM (n = 3). ns, no significant difference. **P < 0.01. (B) PTEN^−/− U2OS cells with reexpression of control S-tag, wild-type PTEN (PTEN-WT), PTEN R159K, PTEN C124S, or PTEN G129E were introduced with Flag-tag control, wild-type PRMT6, methyltransferase-dead PRMT6 E155/164A mutant, or the cancer-derived PRMT6 E155Q mutant. pAKT (S473) and total AKT levels were analyzed. GAPDH served as a loading control.

Fig. 3.

PRMT6 methylates PTEN in vitro and in cells. (A) PRMT6 promotes PTEN dimethylation in vitro. Four micrograms of purified His-PTEN was incubated with 8 μg of purified His-tagged wild-type PRMT6, PRMT6 E155/164A, or PRMT6 E155Q mutant in 50 μL of HMT buffer for 2 h at 37 °C. Methylation of PTEN protein was analyzed by immunoblotting with antibodies including anti-MMA motif (α-MMA), anti-sDMA motif (α-sDMA), and anti-aDMA motif (α-aDMA). Loading of purified His-PTEN and -PRMT6 proteins is shown. (B) PRMT6 asymmetrically dimethylates PTEN in vitro in a dose-dependent manner. Two micrograms of purified His-PTEN was incubated with different amounts of purified His-PRMT6 (0, 2, 4, or 8 μg) in 25 μL of HMT buffer for 2 h at 37 °C. Methylation of PTEN protein was analyzed with immunoblotting with antibodies including anti-MMA motif (α-MMA), anti-sDMA motif (α-sDMA), and anti-aDMA motif (α-aDMA). Loading of purified His-PTEN and -PRMT6 proteins is shown. (C) S-tagged PTEN, PTEN R159K, PTEN C124S, or PTEN G129E was coexpressed with FLAG-tagged PRMT6 in PTEN^−/− U2OS cells and purified with S-protein beads. Purified proteins were analyzed by Western blotting with PTEN R159me2a and PTEN antibodies. Levels of exogenous PRMT6 are shown, and GAPDH served as a loading control. (D) S-tagged PTEN or PTEN R159K was coexpressed with FLAG-tagged wild-type PRMT6 or PRMT6 mutants as indicated in PTEN^−/− U2OS cells and purified with S-protein beads. Purified proteins were analyzed by Western blotting with PTEN R159me2a and PTEN antibodies. Levels of indicated exogenous PRMT6 proteins are shown, and GAPDH served as a loading control. (E) Lysates from PTEN^+/+ and PTEN^−/− U2OS cells were immunoprecipitated with a PTEN mouse monoclonal antibody (sc-7974) and subjected to Western blotting with a PRMT6 antibody. * indicates IgG heavy chain. In E, Left, the expression levels of PTEN and PRMT6 were analyzed, and GAPDH served as a loading control. (F) Cytoplasmic and nuclear extracts from PTEN^+/+ and PTEN^−/− U2OS cells were immunoprecipitated with anti-PTEN agarose resin. The immunoprecipitates were eluted by glycine–HCl (pH 3.5), neutralized with Tris⋅HCl (pH 8.8), and subjected to Western blotting using anti-PTEN, anti-PTEN R159me2a, and anti-PRMT6 antibodies. In F, Left, the expression levels of PTEN and PRMT6 were analyzed. β-tubulin served as a loading control in the cytoplasmic extracts, and HDAC1 served as a loading control in the nuclear extracts.

Fig. 4.

PTEN R159 methylation is important for PTEN tumor-suppressive function. (A) PTEN-null U-87 MG cells expressing S-tag control (Control-Tag), wild-type PTEN (WT-PTEN), PTEN R159K, PTEN C124S, or PTEN G129E yielded tumors (Left). Two nude mice bearing tumors expressing PTEN R159K died significantly earlier than the other nude mice, and their tumors were excluded from statistical analysis. (A, Right) Tumors shown in A, Left were weighed and analyzed. Data were analyzed with the unpaired two-tailed Student’s t test. Error bars indicate SEM. *P < 0.05; **P < 0.01. ns, no significant difference. (B and C) PRMT6-proficient and -deficient U2OS (B) or SF763 (C) cells were injected into symmetrical sites on the back in the same nude mouse. Data were analyzed with the paired two-tailed Student’s t test. Error bars indicate SEM. *P < 0.05.

Fig. 5.

PTEN R159 methylation is involved in regulation of pre-mRNA splicing. (A) Quantification of differentially expressed alternative splicing events in three pairwise comparisons (PTEN vs. control, PTEN R159K vs. PTEN, and PTEN R159K vs. control). A5SS or A3SS, alternative 5′ or 3′ splice site; MXE, mutually exclusive exons; RI, retained introns; SE, skipped exons. Significant events are listed in Datasets S1–S3. (B) Intersections of alternative splicing events among three pairwise comparisons (Left) containing 18 combinatorial modes of change, as listed in Right. Alternative splicing events in 6 of the 18 combinatorial modes account for about 96% of all intersected events and can be categorized into three regulatory modes, including those which are regulated by PTEN in a methylation-related manner (majority in yellow), a methylation-unrelated manner (majority in pink), or a nonmethylation dominant negative manner (majority in cyan). The IncLevelDifference index is important for understanding pairwise comparison, intersection analysis, and the related datasets. When the expression level of the inclusive form of a splicing event in X group is higher or lower than in Y group, then the IncLevelDifference index is, respectively, >0 (+) or <0 (−) in pairwise comparison of X vs. Y. For example, the regulatory pattern of methylation-related includes two conditions: (i) The expression level of the inclusive form of a splicing event is higher in the PTEN group, compared with the control group or PTEN R159K group. In this condition, the splicing event does not show a significant difference in pairwise comparison of PTEN R159K vs. control, the IncLevelDifference index is “+” in pairwise comparison of PTEN vs. control, and is “−” in pairwise comparison of PTEN R159K vs. PTEN. (ii) The expression level of the inclusive form of a splicing event is lower in the PTEN group, compared with the control or PTEN R159K group. In this condition, the splicing event does not show a significant difference in pairwise comparison of PTEN R159K vs. control, the IncLevelDifference index is “−” in pairwise comparison of PTEN vs. control and is “+” in pairwise comparison of PTEN R159K vs. PTEN. (C) Heatmap showing pairwise gene coexpression matrices for 220 known or putative splicing factors in normal brain tissues and tissues adjacent to GBM (Left), PTEN-proficient GBM (Center), and PTEN-deficient GBM (Right).