The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase

Abstract

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

Keywords: BafA1, bafilomycin A1; CRE, cAMP response element; CREB; CREB, cAMP responsive element binding protein; ChIP, chromatin immunoprecipitation; EBSS, Earle's balanced salt solution; ITM2A; ITM2A, integral membrane protein 2A; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 β; MAPK, mitogen-activated protein kinase; MTOR, mechanistic target of rapamycin; PKA; PKA, protein kinase A; SQSTM1, sequestosome 1; TPA, 12-O-tetradecanoylphorbol-13-acetate; autophagy; cAMP, cyclic adenosine monophosphate; tfLC3, tandem fluorescent-tagged LC3; v-ATPase; v-ATPase, vacuolar ATPase..

Figure 1.

The ITM2A promoter is regulated by the PKA-CREB pathway. (A) Schematic diagrams of serial deletion constructs of the ITM2A promoter. The numbers to the left of each construct indicate the distance from the transcription start site (TSS). The predicted cis-elements (GATA, CRE) are indicated, and mutations in GATA or CRE are indicated with X's (left panel). Changed nucleotides in mutant constructs are indicated (right panel). (B) The ITM2A promoter is activated by forskolin treatment. HEK293 cells were transfected with reporter constructs. Twenty-four h after transfection, cells were treated with forskolin for 4 h, and luciferase activity was measured. Relative luciferase activity was normalized to renilla luciferase activity and is represented as a fold increase compared with the control. Experiments were performed in triplicate, and the standard deviation is shown. (C) CRE mutation reduces promoter activity. A luciferase assay was carried out with either wild-type promoter or mutant promoters. pGL2–0.5 wild type versus pGL2–0.5 mutant. *P < 0.05; **P < 0.001. (D) Forskolin induces ITM2A expression. HEK293 cells were treated with forskolin for 4 h, and cell lysates were subject to western blot with anti-ITM2A antibody. The bands were quantified and the fold activation is shown. 0 μM vs. 5 μM. *P < 0.05. (E) Phospho-CREB binds to the ITM2A promoter. HEK293 cells were treated with forskolin and a ChIP assay was performed with either normal IgG antibody or an anti-phospho-CREB antibody. (F) Reduced CREB expression decreased ITM2A expression. HEK293 cells were transfected with either nonspecific (NS) siRNA or CREB siRNA and ITM2A expression was measured by semiquantitative PCR.

Figure 2.

ITM2A localizes in the lysosomes. (A) Overexpressed ITM2A is colocalized with lysosomes. HEK293 cells were transfected with Xpress-ITM2A and cells were stained with anti-ITM2A antibody and anti-LAMP1 antibody (lysosome). The bottom panel depicts enlarged images of areas indicated in the top panel by white boxes. Bars: 10 μm. (B) ITM2A is colocalized with lysosomes by forskolin treatment in HEK293 (upper panel). HEK293 cells were incubated with either mock or forskolin (5 μM, 6 h), and cells were immunostained with anti-ITM2A antibody and anti-LAMP1 antibody. Bars: 10 μm. Top and bottom represent 2 different images of same sample (lower panel). Boxes denote enlarged regions.

Figure 3.

ITM2A expression results in the accumulation of autophagosomes. (A) Elevated level of ITM2A expression increased the LC3B-II/LC3B-I ratio. HEK293 cells stably expressing Xpress-ITM2A were incubated in the presence or absence of BafA1 and the cell lysates were subject to western blot with anti-LC3 antibody and anti-SQSTM1 antibody. LC3B-II/LC3B-I ratios and the level of SQSTM1 (the ratio of SQSTM1/ACTB) are indicated. (B) Overexpressing ITM2A increased the level of LC3B-II and SQSTM1. HEK293 cells were transfected with a plasmid encoding Xpress-ITM2A, and the cell lysates were subject to western blot with the indicated antibody. (C) Overexpressing ITM2A induces autophagosome accumulation in HEK293 cells. HEK293 cells stably expressing GFP-LC3B were transfected with either vector or a plasmid encoding ITM2A. Twenty-four h after transfection, cells were fixed and stained with anti-ITM2A antibody. Control cells were either mock-treated or starved in EBSS medium for 2 h in the presence or absence of BafA1 (100 nM). ITM2A transfected cells were either mock-treated or starved in EBSS medium. Bars: 10 μm. (D) ITM2A expression is closely associated with the autophagosome. HEK293 cells stably expressing GFP-LC3B were transfected with the plasmids encoding Xpress-ITM2A (red). Bars: 10 μm. (E) ITM2A is colocalized with the endogenous LC3. The HEK293 cells were transfected with plasmid encoding Xpress-ITM2A and immunostained with anti-LC3 (green) and anti-Xpress (ITM2A, red). Bars: 10 μm.

Figure 4.

The silencing of ITM2A expression deregulates autophagy (A) Silencing ITM2A deregulates autophagy. HeLa cells were transfected with either nonspecific (NS) siRNA or ITM2A siRNA. Forty-eight h after transfection, cells were starved with EBSS for the indicated times, and the cell lysates were subjected to western blot with the indicated antibodies (left panel). The relative ratio of LC3B-II over LC3B-I (fold) was quantified (right panel). The experiments were repeated 3 times, and representative data are shown. 0 h vs. indicated times. NS siRNA vs. ITM2A siRNA. *P < 0.05; **P < 0.005. (B) Silencing ITM2A interferes with autophagic flux. HeLa cells were transfected with siRNA and cells were incubated in the presence or absence of BafA1 (100 nM, 4 h). The relative ratio of LC3B-II over LC3B-I (fold) and SQSTM1 were quantified (right panel). NS siRNA versus ITM2A siRNA. *P < 0.05. Bars: 10 μm. (C) Silencing ITM2A results in enlarged agglomerations of vesicles. HeLa cells were transfected with siRNA and the mRFP-GFP-LC3B reporter construct (tfLC3) with a time interval (24 h), and the cells were treated with mock, starvation or BafA1 conditions (upper panel). The number of the cells with the aggregates was counted using a fluorescence microscope (N=150 ). Control vector vs. ITM2A siRNA. *P < 0.05; NS significant. Bars: 10 μm. (D) Enlarged vesicles colocalize with LAMP1 protein. HeLa cells were transfected with siRNA and GFP-LC3B with a time interval (24 h), and the cells were immunostained with anti-LAMP1 antibody. Bars: 10 μm.

Figure 5.

ITM2A expression interferes with autophagic flux. (A) HEK293 cells expressing mRFP-GFP-LC3B protein were transfected with either vector or the plasmid encoding ITM2A. Twenty-four h after transfection, cells were fixed and stained with an anti-ITM2A antibody. Control cells were either mock-treated or starved in EBSS medium for 3 h in the presence or absence of BafA1 (100 nM, 4 h). Bars: 10 μm. (B) Quantification of cells with either autophagosomal LC3 puncta or autolysosomal LC3 puncta (N = 300 ). (C) HEK293 cells were transfected with plasmid encoding mRFP-LC3B with either vector (first and second row) or the plasmid encoding ITM2A (third and fourth row), and subjected to either mock-treatment (first and third rows) or starved in EBSS medium (second and fourth rows) for 2 h, and stained with an anti-LAMP1 antibody and an anti-ITM2A antibody. (D) Overexpression of ITM2A increases the level of LC3B. HEK293 cells were transfected with the plasmid encoding GFP-LC3B (0.5 μg) in combination with the plasmid encoding Xpress-ITM2A (0, 0.5, 1 or 1.5 μg). Cell lysates were subject to western blot with the indicated antibodies.

Figure 6.

ITM2A interacts with v-ATPase. (A) Interaction between endogenous ITM2A and endogenous ATP6V0A4. HeLa cells were immunoprecipitated with either normal IgG or an anti-ITM2A antibody, followed by immunoblotting with an anti-ATP6V0A4 antibody (upper and middle panels) or anti-ITM2A antibody (lower panel). WCL, whole cell lysates. (B) Interaction between ITM2A and ATP6V0A4. HEK293 cells were transfected with HA-ITM2A, His-tagged ATP6V0A4 (His-ATP6V0A4) or both. ATP6V0A4 protein was immunopurified with an anti-His antibody and immunoprecipitates were immunoblotted with an anti-ITM2A antibody. (C) Interaction between ITM2A and endogenous ATP6V0A1. HEK293 cells were transfected with HA-ITM2A, and HA-ITM2A was immunopurified to examine the interaction with endogenous ATP6V0A1 (left). Simplified figure of v-ATPase complex is shown and ATP6V0A1, ATP6V0A4, and ATP6V1B1/ATP6V1B2 are indicated (right). (D) ITM2A is colocalized with ATP6V0A4. HEK293 cells were transfected with plasmid encoding Xpress-ATP6V0A4 in the presence or absence of plasmid encoding ITM2A. Cells were mock-treated, starved for 2 h or incubated with BafA1 (100 nM, 4 h) and immunostained with anti-Xpress (red) or anti-ITM2A (green) antibodies. Bars: 10 μm. (E) ITM2A is colocalized with ATP6V0A1. HEK293 cells were transfected with plasmid encoding Xpress-ATP6V0A1 in the presence or absence of plasmid encoding ITM2A. Bars: 10 μm. (F) Schematic diagram of ITM2A and its deletion mutants (N1 to N4, C1). Numbers correspond to the amino acid sequence. TM, transmembrane domain. (G) Identification of the region of ITM2A required for v-ATPase interaction. (i) HEK293 cells were transfected with Xpress-tagged ITM2A mutants (N1, N2, N3, N4), and Xpress-ITM2A mutants were immunopurified with anti-Xpress antibody. The immunoprecipitates were probed with anti- ATP6V0A1 antibody. (ii) HEK293 cells were transfected with Xpress-ITM2A wild type (WT) or Xpress-tagged ITM2A C1 (C1). Xpress-ITM2A proteins were immunopurified with an anti-Xpress antibody and immunoprecipitates were immunoblotted with an anti-ATP6V0A1 antibody.

Figure 7.

ITM2A interferes with lysosomal function. (A) The N-terminal domain of ITM2A is required for autophagosome formation. ITM2A deletion mutants were expressed in GFP-LC3B cells and stained with anti-Xpress antibody. Bars: 10 μm. (B) The number of GFP-LC3B dot-positive cells (N > 5) was counted using a fluorescent microscope (N=300 ). Control vector versus ITM2A mutant. *P < 0.001; **P < 0.005; NS, not significant. (C) ITM2A C1 did not increase the LC3B-II/LC3B-I ratio. HEK293 cells were transfected with vector, ITM2A WT, or ITM2A C1 and the cell lysates were subjected to western blot with anti-LC3 antibody. (D) ITM2A interferes with the acidification of lysosomes. GFP-LC3B cells were transfected with plasmid encoding ITM2A. Twenty-four h after transfection, cells were starved for 3 h and stained with LysoTracker Red dye (left panel). The number of LysoTracker Red spots was counted using a fluorescence microscope (right panel). N = 50 , *P < 10^–19; NS, not significant. Bars: 10 μm. (E) Lysosomal pH values were measured using LysoSensor Yellow/Blue-dextran. N = 3 , *P < 0.05 ; NS, not significant.

Figure 8.

A proposed hypothesis of the cellular pathway involving ITM2A and v-ATPase regulating the acidification of autophagosomes and autophagic flux. (A) v-ATPase is involved in the transport of hydrogen ion, and also involved in the fusion of autophagosomes with lysosomes. (B) Upregulation of ITM2A expression inhibits the transport of hydrogen ion by interacting with the v-ATPase. Inactivation of the v-ATPase inhibits the fusion of autophagosomes with lysosomes, and contributes to the accumulation of autophagosomes and lysosomes.