Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Mar 28;97(7):3028-33.
doi: 10.1073/pnas.97.7.3028.

Tcl1 enhances Akt kinase activity and mediates its nuclear translocation

Y Pekarsky  1 , A Koval, C Hallas, R Bichi, M Tresini, S Malstrom, G Russo, P Tsichlis, C M Croce
Affiliations

Tcl1 enhances Akt kinase activity and mediates its nuclear translocation

Y Pekarsky et al. Proc Natl Acad Sci U S A. .

Abstract

The TCL1 oncogene at 14q32.1 is involved in the development of human mature T-cell leukemia. The mechanism of action of Tcl1 is unknown. Because the virus containing the v-akt oncogene causes T-cell lymphoma in mice and Akt is a key player in transduction of antiapoptotic and proliferative signals in T-cells, we investigated whether Akt and Tcl1 function in the same pathway. Coimmunoprecipitation experiments showed that endogenous Akt1 and Tcl1 physically interact in the T-cell leukemia cell line SupT11; both proteins also interact when cotransfected into 293 cells. Using several AKT1 constructs in cotransfection experiments, we determined that this interaction occurs through the pleckstrin homology domain of the Akt1 protein. We further demonstrated that, in 293 cells transfected with TCL1, the endogenous Akt1 bound to Tcl1 is 5-10 times more active compared with Akt1 not bound to Tcl1. The intracellular localization of Tcl1 and Akt1 in mouse fibroblasts was investigated by immunofluorescence. When transfected alone, Akt1 was found only in cytoplasm whereas Tcl1 was localized in the cytoplasm and in the nucleus. Interestingly, Akt1 was also found in the nucleus when AKT1 was cotransfected with TCL1, suggesting that Tcl1 promotes the transport of Akt1 to the nucleus. These findings were supported by the intracellular localization of Akt1 or Tcl1 when Tcl1 or Akt1, respectively, were confined to the specific cellular compartments. Thus, we demonstrate that Tcl1 is a cofactor of Akt1 that enhances Akt1 kinase activity and promotes its nuclear transport.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Tcl1 interacts with Akt. (A) Immunoprecipitation of Akt1 with anti-Tcl1 antibody. Detection of Akt1 in the immunoprecipitates was carried out by Western blotting using a mouse monoclonal anti-Akt1 antibody. Lysates used were 293 cells transfected with TCL1 (lanes 1–3) and SupT11 cells (lanes 4–6). Antibodies used for immunoprecipitation were anti-Tcl1 (lanes 1 and 4), mouse IgG (lanes 2 and 5), and mouse monoclonal anti-Akt1 (lanes 3 and 6). (B) Akt1 interacts with Tcl1 through PH domain. 293 cells were cotransfected with TCL1 and HA-AKT1 or HA-(Δ11-60) AKT1 mutant as indicated. Immunoprecipitations were carried out with an anti-HA antibody (lanes 1 and 2), mouse IgG (lanes 3 and 4), or anti-Tcl1 antibody (lanes 5 and 6) and were detected by Western blotting with anti-Tcl1 antibody. Lanes 7 and 8: The lysate was coprecipitated with 5 μg of Akt1 PH domain-GST fusion protein (lane 7) or GST alone (lane 8). (C) Akt1 but not Akt2 strongly interacts with Tcl1. 293 cells were cotransfected with TCL1 and HA-AKT1 (lanes 1–3) or HA-AKT2 (lanes 4–6). Immunoprecipitations were carried out with anti-Tcl1 antibody (lanes 1 and 4), mouse IgG (lanes 2 and 5), and anti-HA antibody (lanes 3 and 6) and were detected with anti-Tcl1 antibody. (D) Interaction with Tcl1 is independent of Akt1 phosphorylation. 293 cells were cotransfected with TCL1 and HA-AKT1 (lanes 1–3) or HA-AKT1 AA mutant (Thr308/Ala Ser473/Ala). Immunoprecipitations and Western blot detection were performed as in C. Expression levels of exogenous and endogenous Tcl1 and Akt were checked in each experiment (where applicable) and were found similar.
Figure 2
Figure 2
Tcl1 enhances Akt1 kinase activity. Endogenous Akt1 was immunoprecipitated from 293 cells transfected with the indicated constructs. Kinase activity was determined by using GSK3-β-GST fusion protein as a substrate. Each reaction was terminated after 0, 4, 10, and 30 min. Amounts of Akt (upper panels) and phospho-GSK3-β (lower panels) were determined by Western blotting with rabbit anti-Akt antibody and anti-phospho-GSK3-β antibody, respectively. (A) Akt1 was immunoprecipitated from TCL1-transfected cells with a anti-Tcl1 antibody (Left) or vector-transfected cells with anti-Akt antibody (Right). (B) Same lysates as in A, but immunoprecipitations were carried out with an anti-Akt antibody only. (C) Lysates of thymus from a TCL1-transgenic mouse (2) (left two lanes) or a wild-type mouse (right two lanes) were immunoprecipitated with anti-Akt antibody. For immunoprecipitation of Akt, we used anti-PKBα/Akt1 clone 7 antibody, anti-Akt/PKB rabbit polyclonal antibody, or anti-Akt antibody included with Akt kinase assay kit, with consistent results.
Figure 3
Figure 3
The expression of Tcl1 does not increase Akt1 phosphorylation or interfere with effect of wortmannin. NIH 3T3 cells were transfected with TCL1 (lanes 1, 3, 5, and 7) or vector (lanes 2, 4, 6, and 8) and were starved with media without FCS overnight. (A) Cells were treated with 100 ng/ml PDGF for the indicated period of time and were lysed. Western blotting was performed by using anti-phospho-Akt and anti-Tcl1 antibody. Each lane contains the same amount of protein (not shown). (B) NIH 3T3 were transfected and starved as in A. Cells were not treated (lanes 1 and 2); were treated with 200 nM wortmannin for 1.5 hours (lanes 3 and 4); were treated with 100 ng/ml PDGF for 30 min (lanes 5 and 6); and were treated with 200 nM wortmannin for 1 hour followed by PDGF for 30 min (lanes 7 and 8). Western blotting was performed as in A.
Figure 4
Figure 4
Tcl1 promotes nuclear translocation of Akt1. MEF cells were transfected or cotransfected with indicated constructs. (A) Intracellular localization of Akt1 (Left), Tcl1 (Center), and GFP-Tcl1 (Right). (B) Colocalization of Akt1 (green) and Tcl1 (red). (C) Colocalization of Akt1 (red) and GFP-Tcl1 (green). (D) Intracellular localization of Akt1 (red) and Tcl1-GFP (green).
Figure 5
Figure 5
Nuclear translocation of Akt1 by Tcl1 require their interaction in the cytoplasm. MEF cells were transfected or cotransfected with indicated constructs. (A) Intracellular localization of Akt1 (red) and nuc-Tcl1 (green) in the same cells. (B) Intracellular localization of myristoylated Akt1 (green) and Tcl1 (red) in the same cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Virgilio L, Narducci M G, Isobe M, Billips L G, Cooper M D, Croce C M, Russo G. Proc Natl Acad Sci USA. 1994;91:12530–12534. - PMC - PubMed
    1. Virgilio L, Lazzeri C, Bichi R, Nibu K, Narducci M G, Russo G, Rothstein J L, Croce C M. Proc Natl Acad Sci USA. 1998;95:3885–3889. - PMC - PubMed
    1. Soulier J, Madani A, Cacheux V, Rosenzwajg M, Sigaux F, Stern M H. Oncogene. 1994;9:3565–3570. - PubMed
    1. Pekarsky Y, Hallas C, Isobe M, Russo G, Croce C M. Proc Natl Acad Sci USA. 1999;96:2949–2951. - PMC - PubMed
    1. Hallas C, Pekarsky Y, Itoyama T, Varnum J, Bichi R, Rothstein J, Croce C M. Proc Natl Acad Sci USA. 1999;96:14418–14423. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources