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. 2013 Oct 1;12(19):3165-74.
doi: 10.4161/cc.26183. Epub 2013 Aug 27.

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle (V体育安卓版)

Guillermo Risso  1 , Federico Pelisch, Berta Pozzi, Pablo Mammi, Matías Blaustein, Alejandro Colman-Lerner, Anabella Srebrow
Affiliations

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle

Guillermo Risso (V体育安卓版) et al. Cell Cycle. .

Abstract

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase VSports手机版. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G₁/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase. .

Keywords: Akt/PKB; SUMO; alternative splicing; cell cycle; post-translational modification; signal transduction. V体育安卓版.

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"VSports在线直播" Figures

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Figure 1. Akt1 is a SUMO substrate in cells and in vitro. (A) HEK 293T cells were co-transfected with expression vectors for HA-tagged Akt1 and His-SUMO2, with or without T7-Ubc9 expression vector. Cell lysates were prepared 48 h after transfection and analyzed by SDS/page and western blot with an anti-HA antibody. Slower migrating bands suspected to be SUMO-Akt1 conjugates are indicated with an asterisk. (B) HEK 293T cells were transfected with HA-Akt1 and Ubc9 expression vectors, with or without His-SUMO2. After 48 h, cells were lysed, an aliquot of the lysate was taken as input, and the reminder was subject to denaturing Ni2+ affinity chromatography. Both fractions were analyzed by western blot with an anti-HA antibody. Different lanes from the same gel were put together as indicated by the dividing line. (C) Cells were co-transfected with HA-Akt1, His-SUMO2, Ubc9 and either SENP1 or 2 expression vectors, or the corresponding empty vector (−). SUMO conjugation to Akt1 was analyzed as in (B). (D) Pull-down assay (right panel) was performed combining bacterially expressed and purified GST or GST-Ubc9 with cell lysates from HEK 293T expressing HA-Akt1. An aliquot of the lysate was taken as input (left panel). Samples were analyzed by western blot with an anti-HA antibody. (E) Purified recombinant HA-Akt1 was incubated with Aos1-Uba2 (E1 heterodimer), increasing amounts of Ubc9 (E2 enzyme), and SUMO2. Reactions were stopped by addition of 2X Laemmli sample buffer and analyzed by western blot with an anti-HA antibody.
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Figure 2. Mutational analysis of Akt1 SUMO conjugation sites. (A) HEK 293T cells were transfected either with wild-type (WT), K276R, K301R, or the double mutant K276R/K301R (“2KR”) HA-tagged Akt1 expression vectors, together with 6xHis-tagged SUMO2 and Ubc9 expression vectors. Cells lysates were subject to denaturing Ni2+ affinity chromatography and analyzed by western blot with an anti-HA antibody as indicated in Figure 1. Different lanes from the same gel were put together as indicated by the dividing lines. (B) Analysis of the SUMO consensus site surrounding K276 in Akt1. CM, canonical consensus motif; ICM, inverted consensus motif; Ψ, bulky, hydrophobic amino acid; X, any amino acid; E, glutamic acid; D, aspartic acid. (C) HEK 293T cells were transfected either with wild-type, K276R, or the double mutant D274N/E278Q HA-tagged Akt1 expression vectors, together with 6xHis-tagged SUMO2 and Ubc9. Cell lysates were subject to denaturing Ni2+ affinity chromatography and analyzed by western blot with an anti-HA antibody as indicated above. Different lanes from the same gel were put together as indicated by the dividing line.
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Figure 3. Lack of subordination between phosphorylation and SUMOylation of Akt1. (A) HEK 293T cells were transfected either with wild-type or 2KR HA-tagged Akt1 expression vectors and cultured with 10% fetal bovine serum (FBS) or without serum (0% FBS). Cells lysates were subject to immunoprecipitation with anti-HA antibody and analyzed by western blot with the antibodies indicated on the right side of each panel. (B) HEK 293T cells were transfected with HA-Akt1 and 6xHis-tagged SUMO2, with or without Ubc9 expression vectors, as indicated at the top of the panel. Twenty-four h after transfection cells were treated with the Pi3K inhibitor LY 294002 (LY) for another 24 h before cell lysis. An aliquot of the lysates was taken as input and the reminder was subject to denaturing Ni2+ affinity chromatography. Both fractions were analyzed by western blot with the antibodies indicated at the bottom of each panel. (C) HEK 293T cells were transfected either with wild-type, T308D/S473D, or T308A HA-tagged Akt1, 6xHis-SUMO2, and Ubc9 expression vectors. An aliquot of the lysates was taken as input and the reminder was subject to denaturing Ni2+ affinity chromatography. Both fractions were analyzed by western blot with anti-HA antibody.
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Figure 4. Akt1 SUMOylation affects alternative splicing patterns. (A) Partial representation of fibronectin (FN EDI) splicing reporter minigene used to analyzed alternative splicing patterns in transfected cells. The scheme shows the 2 mRNA isoforms, either containing (EDI+) or lacking (EDI-) EDI alternative exon, derived from a single pre-mRNA upon transcription of the minigene. (B) HeLa cells were co-transfected with the FN EDI minigene, either with wild-type (WT), 2KR HA-tagged Akt1 expression vectors, or the corresponding empty vector (−), and with an expression vector for a constitutively active form of Ras (RasV12C40) or its corresponding empty vector. Forty-eight h after transfection, RNA was prepared and used to analyze mRNA isoforms derived from the minigene by reverse transcription and radioactive PCR with specific primers. The bar graph corresponds to the ratio between the 2 PCR products shown in the lower panel. The different EDI+/EDI- ratios were standardized against the ratio corresponding to the condition lacking both Akt and Ras isoforms. Different lanes from the same gel are shown. (C) HeLa cells were transfected with siRNA against Akt1 3′UTR (siAkt1) or control siRNA (−). Cells were re-transfected 24 h later with the FN EDI minigene and increasing amounts of HA-Akt1 WT, HA-Akt1 2KR, or the corresponding empty vector (−) and cultured for additional 48 h. EDI splicing patterns were analyzed as indicated in (B). The bar graph corresponds to the ratio between the two PCR products. (D) HeLa cells were transfected either with WT, E17K, or E17K/2KR HA-tagged Akt1 expression vectors together with 6xHis-SUMO2 and Ubc9 expression vectors. An aliquot of the lysates was taken as input and the reminder was subject to denaturing Ni2+ affinity chromatography. Both fractions were analyzed by western blot with anti-HA antibody. Different lanes from the same gel were put together as indicated by the dividing line. (E) HeLa cells were transfected with the FN EDI minigene and either WT, 2KR, E17K, or E17K/2KR HA-Akt1 expression vectors or the corresponding empty vector. Splicing isoforms derived from the minigene were analyzed as in (B). (F) Partial, schematic representation of the Bcl-x splicing reporter minigene and the 2 mRNA isoforms, long (L) and short (S), derived from it. (G) HEK 293T cells were transfected with the Bcl-x splicing reporter minigene together with WT or 2KR HA-Akt1 expression vectors, or the corresponding empty vector. Forty-eight h later, RNA was extracted and mRNA isoforms derived from the transfected minigene were analyzed by reverse transcription and radioactive PCR with specific primers. The bar graph shows the ratio between the PCR products corresponding to the long [Bcl-x(L)] and the short [Bcl-x(S)] mRNA isoforms. In every case, values shown in histograms correspond to mean ± SE of a representative experiment run in duplicate.
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Figure 5. SUMOylation of Akt1 affects cell cycle progression. HEK 293T cells were transfected with empty vector (control), WT or 2KR HA-Akt1 expression vectors and cell cycle profiles (DNA content) were analyzed by flow cytometry 48 h after transfection. The bar graph shows the percentage of cells at the different cell cycle phases for each analyzed condition.
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