Cellular transformation by the MSP58 oncogene is inhibited by its physical interaction with the PTEN tumor suppressor - PubMed (original) (raw)

Cellular transformation by the MSP58 oncogene is inhibited by its physical interaction with the PTEN tumor suppressor

Koichi Okumura et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The PTEN (phosphatase and tensin homologue) tumor suppressor protein contains a single catalytic domain with both lipid and protein phosphatase activities. The remaining C-terminal half of the PTEN protein plays a role in its stability and is mutated in many clinical cancer samples. Here, we report that the PTEN C-terminal domain physically interacts with the forkhead-associated domain of the oncogenic MSP58 protein and that this interaction requires PTEN Thr-366. We further show that while MSP58 transforms Pten-/- mouse embryo fibroblasts (MEFs), concurrent introduction of wild-type PTEN causes a dramatic reduction in the number of MSP58-induced transformed foci. This PTEN-mediated inhibition of cellular transformation requires physical interaction as evidenced by the failure of PTEN(T366A) point mutation (residing within the MSP58 interaction domain) to suppress MSP-58-driven transformation. These observations, together with the capacity of catalytically inactive PTEN mutant (G129R) to suppress MSP58 oncogenicity, support the view that the C-terminal region of PTEN directly provides a previously uncharacterized biological function in its ability to regulate cellular transformation.

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Figures

Fig. 1.

Fig. 1.

PTEN interacts with MSP58. (A) 293T cell lysates were precipitated with anti-PTEN or control IgG antibody followed by immunoblotting for coprecipitated MSP58. (B) Similarly, lysates were precipitated with anti-MSP58 followed by immunoblotting for coprecipitated PTEN. Efficiency of immunoprecipitation was assessed by reprobing each blot with antibodies used for precipitation. (C) 35S-labeled MSP58 protein, produced by TNT T7-coupled reticulocyte lysate, was applied to GST or GST-PTEN prebound to glutathione Sepharose (GST) beads for pull-down assays. (D) Lysates of 293T cells, which were transfected with empty vector or Flag-MSP58 plasmid, were applied to GST beads for pull-down assays. The pull-down product was detected by immunoblotting with anti-Flag tag horseradish peroxidase (HRP)-conjugated antibody. (E) Immunostaining for PTEN and MSP58 MEFs wild-type or null for PTEN.

Fig. 2.

Fig. 2.

MSP58 binds to the C terminus of PTEN through the MSP58 FHA domain. Lysates of 293T cells, transfected with various combinations of GFP-tagged (PTEN, full-length, amino acids 1–403; NPTEN, amino acids 1–202; PTENC, amino acids 186–403) and Flag-tagged (MSP58, full-length, amino acids 1–462; N-MSP58, amino acids 1–290; MSP58-C, amino acids 290–462) expression plasmids, were used in a coimmunoprecipitation (IP) assay. (A) Lysates of 293T cells were subjected to coimmunoprecipitation with anti-GFP antibody-conjugated agarose beads. Coimmunoprecipitated products were detected by immunoblotting with anti-Flag antibody. For confirmation of IP efficiency, the membrane was stripped and reprobed with anti-GFP antibody. (B) Lysates were incubated with anti-Flag antibody-conjugated agarose. Coimmunoprecipitated products were detected by immunoblotting with anti-GFP horseradish peroxidase (HRP)-conjugated antibody. For confirmation of IP efficiency, the membrane was stripped and reprobed with anti-Flag HRP-conjugated antibody.

Fig. 3.

Fig. 3.

Thr-366 of PTEN is critical for binding to MSP58. (A) Schematic diagram of phosphorylation sites in the PTEN C-terminal region and the FHA domains of various proteins displaying similarity to PTEN. (B) Lysates of 293T cells, cotransfected with MSP58 and various PTEN mutant expression plasmids, were used in a coimmunoprecipitation (IP) assay. Lysates were incubated with anti-Flag antibody conjugated agarose. Coimmunoprecipitated products were detected by immunoblotting with anti-GFP horseradish peroxidase-conjugated antibody. (Lower) Immunoblotting of GFP PTEN mutants for expression control.

Fig. 4.

Fig. 4.

PTEN interaction inhibits focus formation by MSP58. _Pten_–/– MEFs were infected with viruses expressing the indicated proteins. The cells were incubated for 40 days and then fixed and stained with crystal violet.

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