Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8

Abstract

The myotubularins are a large family of inositol polyphosphate 3-phosphatases that, despite having common substrates, subsume unique functions in cells that are disparate. The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] at the D-3 position. Seven members are inactive because they lack the conserved cysteine residue in the CX(5)R motif required for activity. We studied a subfamily of homologous myotubularins, including myotubularin-related protein 6 (MTMR6), MTMR7, and MTMR8, all of which dimerize with the catalytically inactive MTMR9. Complex formation between the active myotubularins and MTMR9 increases their catalytic activity and alters their substrate specificity, wherein the MTMR6/R9 complex prefers PtdIns(3,5)P(2) as substrate; the MTMR8/R9 complex prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of MTMR6 toward PtdIns(3,5)P(2) by over 30-fold, and enhanced the activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 increased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns(3,5)P(2) and PtdIns(3)P, respectively. In cells, the MTMR6/R9 complex significantly increases the cellular levels of PtdIns(5)P, the product of PI(3,5)P(2) dephosphorylation, whereas the MTMR8/R9 complex reduces cellular PtdIns(3)P levels. Consequentially, the MTMR6/R9 complex serves to inhibit stress-induced apoptosis and the MTMR8/R9 complex inhibits autophagy.

Fig. 1.

Interaction between MTMR8 and MTMR9. (A) HeLa cells were cotransfected with HA-MTMR8 and MTMR9-FLAG for 24 h, and proteins were immunoprecipitated with anti-HA and anti-FLAG polyclonal antibodies. Rabbit IgG was used as a negative control. Western blotting was done using monoclonal antibodies against HA (Upper) or FLAG (Lower) tags. (B) Stability of MTMR8 is increased by the formation of the complex. HeLa cells were transfected with HA-MTMR8, or HA-MTMR8 plus FLAG-MTMR9 for 24 h. Cycloheximide (150 μg/mL) was added for the times indicated. The levels of MTMR8 and MTMR9 were detected by Western blotting with an anti-HA or anti-FLAG antibody, respectively. β-Actin levels are shown as a loading control. MTMR8 band intensities relative to actin are indicated below each lane.

Fig. 2.

Enzymatic activity of MTMR6, MTMR6/R9, MTMR8, and MTMR8/R9 toward PtdIns(3)P (A) or PtdIns(3,5)P₂ (B). (C) Measurement of cellular PtdIns(5)P levels. HeLa cells were transfected with vector (control), cotransfected with either MTMR6 plus MTMR9 or MTMR8 plus MTMR9, and PtdIns(5)P levels were measured as previously described (45). Results are presented as relative mass compared with that in vector-transfected cells. (D) Overexpression of MTMR8 plus MTMR9 reduces levels of PtdIns(3)P in COS-7 cells. Cells were stained with anti-PI(3)P antibodies followed by anti-mouse Alexa568-conjugated antibodies. Nuclei are visualized with DAPI. Cotransfection efficiency was determined in a separate set of plates, because of limited available fluorescence channels, and was found to be greater that 95% in all cases. (Magnification: 63×.) (E) Quantification of immunofluorescence shown in D, expressed as an average number of PI(3)P spots per cell normalized to the number seen in MTMR8/R9 expressing cells. (F) Overexpression of MTMR8/R9 has no effect on apoptosis. HeLa cells were treated with the indicated constructs for 36 h, then with 100 µM etoposide to induce apoptosis. After 8 h, cells were treated with APOPercentage dye to selectively stain apoptotic cells with dark spots. Percentage of apoptotic cells are counted as the number of darker cells out of every 100 cells (*P < 0.01, t test).

Fig. 3.

The MTMR8/R9 complex regulates autophagy. (A) HeLa cells were transfected with the indicated constructs. Western blotting was used to detect MTMR8, MTMR9, and p62. A nonspecific band (*) is seen below MTMR9 in the vector and MTMR8 lanes. (B) Western blotting of p62 in HeLa cell extracts transfected with the indicated RNAi constructs. (C) HeLa cells were transfected for 24 h with the indicated RNAis, then treated for 4 h with 100 nM Bafilomycin A1 (BafA1), 10 µg/mL E64d, and 10 µg/mL pepstatin. Cell lysates were immunoblotted with anti-p62 and anti–β-actin, and p62 band intensities relative to actin are indicated below each lane. (D) HeLa cells were transfected with HA-MTMR8 for 24 h, treated with the indicated RNAi constructs for another 24 h, then analyzed by blotting with an antibody against HA tag. (E) The MTMR8 and MTMR9 complex dissociates during starvation induced autophagy. HeLa cells were transfected with HA-MTMR8 for 36 h, followed by serum starvation for the indicated times. Extracts were immunoprecipitated with an antibody against MTMR9 and immunoblotted with an anti-HA antibody to detect MTMR8. The protein levels of MTMR8 and MTMR9 during starvation are shown in input panels.

Fig. 4.

Knockdown of the MTMR8/R9 complex induces autophagy. (A) HeLa cells were transfected with GFP-WIPI-1 for 24 h, then treated with indicated RNAi constructs for 24 h. Autophagy was assessed by measuring WIPI-1 puncta-formation by immunofluorescence. (Magnification: 63×.) (B) Results from a total of 500 cells were counted and the ratios of cells in puncta/nonpuncta status was determined. (*P < 0.01.) (C) Overexpression of the MTMR8/R9 complex suppresses autophagy. HeLa cells were transfected with GFP-WIPI-1 for 24 h, then transfected with the indicated constructs for an additional 24 h. Autophagy was induced by rapamycine for 3 h and then was measured by immunofluorescence. (Magnification: 63×.) (D) The ratios of cells in puncta/nonpuncta status was measured. Cotransfection efficiency was determined to be greater than 95% using a duplicate set of plates. (E) The hypothesized consequential effects of complex formation between MTMR9 and either MTMR8 or MTMR6 are shown in a schematic diagram.