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First published online 1 June 2004
doi: 10.1242/jcs.01142

Journal of Cell Science 117, 3021-3029 (2004)
Published by The Company of Biologists 2004
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Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation

Luguo Sun1, Lanying Liu1,*, Xiang-Jiao Yang2 and Zhenguo Wu1,{ddagger}

1 Department of Biochemistry, Hong Kong University of Science and Technology, Hong Kong, China
2 Molecular Oncology Group, Department of Medicine, McGill University Health Center, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1, Canada



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  		Fig. 1. PHB2 specifically interacts with Akt. (A) Full-length PHB2 and the truncated PHB2 found in the yeast two-hybrid screen. The numbers represent the positions of amino acids. Abbreviation: TM, potential transmembrane domain. (B) Cos-7 cells were co-transfected with xp-PHB2 together with either HA-Akt or HA-MKK6. (C) Cell lysates containing xp-PHB2 were mixed with C2C12 WCEs harvested before and after differentiation. Immunoprecipitation and immunoblot were carried out as indicated. Abbreviations: CoIP, co-immunoprecipitation; GM, growth medium; IP, immunoprecipitation; IB, immunoblot; SF, serum-free medium; Xp, Xpress tag. Input represents 10% of total lysates used for immunoprecipitation.

 


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  		Fig. 2. Further localization of interaction domains on both PHB2 and Akt. Different deletion fragments of the PHB2 (A) or Akt (B) gene were separately cloned into pGADT7 and pGBKT7, respectively. The resulting clones were tested for their mutual interaction using yeast two-hybrid assays. The black bar represents the Akt-interacting region on PHB2. Abbreviations: CT, C terminus; PH, pleckstrinhomology domain. The plus and minus signs denote growth and no growth, respectively, on SD/-his/-ade/-leu/-trp plates. The numbers represent the position of amino acids in either PHB2 or Akt.

 


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  		Fig. 3. PHB2 represses the transcriptional activity of MyoD and MEF2. C2C12 cells were separately co-transfected with 3xMEF2-luc (A), 4RE-luc (B) or gal4-luc (C), together with various expression vectors as indicated. After 36 hours of growth in GM and 24 hours in DM, cells were harvested and the luciferase activity in each sample was determined. Fold change was calculated as the ratio of the luciferase activity in samples transfected with either PHB2 (A,B) or Gal4MEF2/Gal4-MyoD with or without PHB2 (C) to that of samples transfected with either an empty vector (A,B) or Gal4DBD (C). Abbreviations: DBD, DNA-binding domain (amino acids 1-147 of Gal4); vec, an empty vector. All experiments were done independently three times and the results were presented as mean±s.d.

 


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  		Fig. 4. PHB2 interacts with both MyoD and MEF2 in cells. (A) Cos-7 cells were co-transfected with various expression vectors as indicated. (B) Equal amounts of Cos-7 cell lysates expressing xp-PHB2 were separately incubated with C2C12 cell lysates harvested before and after differentiation. Xp-PHB2 was immunoprecipitated and the co-precipitated MyoD and MEF2 were detected by immunoblot. Abbreviations: GM, growth medium; SF, serum-free medium. Input represents 10% of total lysates used for immunoprecipitation. (C) Yeast AH109 cells were transformed with expression vectors as indicated. The plus and minus signs denote growth and no growth, respectively, on SD/-his/-ade/-trp/-leu plates. AD, pGADT7 vector encoding the Gal4 transcription-activation domain; BD, pGBKT7 vector encoding the Gal4 DNA binding domain.

 


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  		Fig. 5. Stable expression of PHB2 in C2C12 inhibits muscle differentiation. Near-confluent stable C2C12 cells expressing either an empty vector or PHB2 were allowed to differentiate in DM for the indicated times. (A) Cells were harvested and 30 µg WCEs were resolved by SDS-PAGE and subjected to immunoblotting. (B) After 24 hours in DM, cells were fixed, subjected to immunostaining and microscopic imaging. Abbreviations: DM, differentiation medium; GM, growth medium; MHC, myosin heavy chain; Phase, phase contrast.

 


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  		Fig. 6. PHB2 specifically interacts with HDAC1 in cells. (A) Cos-7 cells were co-transfected with various expression vectors as indicated. Either PHB2 or JNKK2 (control) were first immunoprecipitated with the anti-Xpress antibody and the co-precipitated HDAC1 was detected by immunoblot with the anti-Flag antibody. Input represents 10% of total lysates used in the immunoprecipitation. (B) C2C12 cells were co-transfected with PHB2-GFP and Flag-HDAC1. PHB2 was visualized by autofluorescence (green) of GFP and HDAC1 was visualized by indirect immunofluorescence (red) with the anti-Flag antibody. Arrows indicated cells containing both PHB2 and HDAC1. Abbreviations: GFP, green fluorescent protein. Notice the two PHB2-positive cells at the bottom left corner without HDAC1 showing predominant mitochondrial staining.

 


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  		Fig. 7. Akt relieves repressive effect of PHB2 and reduces PHB2 binding to MyoD. (A) C2C12 cells were co-transfected with myogenic reporters together with an empty vector, PHB2 and Aktca in various combinations as indicated. After 36 hours of growth in GM and 24 hours in DM, cells were harvested and the luciferase activity determined. The experiments were performed independently three times with similar results. The results from a representative experiment are presented. (B) C2C12 cells were co-transfected with MLC-lacZ together with either an empty vector or PHB2. At the start of differentiation, either DMSO (vehicle) or IGF1 (100 ng ml-1) were added to DM. After 24 hours, cells were fixed and stained with X-Gal. The numbers on top of each bar denote fold changes in either the luciferase activity (A) or the number of X-gal-positive cells (B) of a sample versus that of the controls. (C) Cos-7 cells were cotransfected with various expression vectors as indicated. PHB2 was immunoprecipitated by the anti-Xpress antibody and the co-precipitated Flag-MyoD and HA-Akt were detected by immunoblot. The expression levels of Flag-MyoD, Flag-Six1, HA-Akt, HAAktkm and HA-MKK6 are shown in the bottom two panels. The identities of the bands on each panel are indicated next to arrows. Abbreviations: ca, constitutively active; km, mutation of lysine 179 to methionine; MLC, myosin light chain; vec, vector.

 

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© The Company of Biologists Ltd 2004