Thymosin beta 4 expression and nuclear transport are regulated by hMLH1

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Abstract

For hMLH1, a key enzyme of DNA mismatch repair and frequently mutated in human cancers, several additional functions have been suggested. We now identified Thymosin β4 (Tβ4), an actin-binding and cell motility regulating protein, by bacterial two-hybrid screening. Interaction was confirmed by coimmunoprecipitation. Tβ4 was weakly expressed in the hMLH1-deficient cell lines 293T and HCT-116. Reconstitution of hMLH1 resulted in strong expression of Tβ4. Confocal laser microscopy revealed nuclear colocalization of both proteins. Reconstitution with hMLH1 mutants lacking a functional nuclear localization sequence resulted in cytoplasmatic retention of both proteins. After 4− or hMLH1-siRNA treatment, cell migration of hMLH1-proficient cells was markedly decreased.

Our results show that hMLH1 interacts with Tβ4 and regulates its expression and nuclear transport. Moreover, loss of hMLH1 causes Tβ4 deprivation and results in reduced migratory activity in vitro. These data give insight into novel functions of hMLH1 and probably disease related dysregulated mechanisms.

Section snippets

Materials and methods

Bacterial Strains and cell lines. E. coli strains XL1-Blue MRF´ Kan, BacterioMatch Two-Hybrid reporter, and validation cells were purchased from Stratagene (La Jolla, CA). All media were prepared as described in the manufacturer’s protocol. HCT-116 cells deficient in hMLH1 (ATCC CCL247) as well as 293T cells, obtained from Dr. Kurt Ballmer (Paul Scherer Institute, Villingen, Switzerland), were grown in DMEM with 10% FCS. As previously published, MutLα is not expressed in 293T [20]. Stably

Results and discussion

During the last years several hMLH1-interacting partners have been identified by yeast two-hybrid. To circumvent the limitations inherent to the yeast system, like delicate identification of nuclear proteins, we applied a bacterial two-hybrid screening. We now report the identification of Tβ4, an actin-binding, and cell motility regulating protein [27], [28]. The physical interaction of hMLH1 with Tβ4 was verified by coimmunoprecipitation in a mammalian system (Fig. 1).

hMLH1 contains a

Acknowledgments

We thank Dr. Françoise Praz for donating the HCT-116 mlh 0–1 and HCT-116 mlh 1–2 cell line and Nicole Weber for performance of experiments.

References (39)

  • T. Huff et al.

    beta-Thymosins, small acidic peptides with multiple functions

    Int. J. Biochem. Cell. Biol.

    (2001)
  • A.L. Goldstein et al.

    Thymosin beta4: actin-sequestering protein moonlights to repair injured tissues

    Trends Mol. Med.

    (2005)
  • T. Kobayashi et al.

    Thymosin-beta4 regulates motility and metastasis of malignant mouse fibrosarcoma cells

    Am. J. Pathol.

    (2002)
  • R. Sankila et al.

    Better survival rates in patients with MLH1-associated hereditary colorectal cancer

    Gastroenterology

    (1996)
  • P. Modrich

    Mechanisms and biological effects of mismatch repair

    Annu. Rev. Genet.

    (1991)
  • E. Cannavo et al.

    Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair

    Cancer Res.

    (2005)
  • G. Plotz et al.

    hMutSalpha forms an ATP-dependent complex with hMutLalpha and hMutLbeta on DNA

    Nucleic Acids Res.

    (2002)
  • H.T. Lynch et al.

    Hereditary colorectal cancer

    N. Engl. J. Med.

    (2003)
  • P. Peltomaki et al.

    Mutations associated with HNPCC predisposition—update of ICG-HNPCC/INSiGHT mutation database

    Dis. Markers

    (2004)
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    This study was supported by a grant from the Wilhelm Sander-Foundation (2005.085.1).

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