The tumour suppressor protein LIMD1 specifically binds the HIF-prolyl hydroxylases and pVHL and represses HIF-1 mediated transcription


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Thomas M Webb1, Thilo Hagen1, Daniel E Foxler1, Maureen Mee1, Yunfeng Feng2, Gregory D Longmore2, Tyson V Sharp1

1University of Nottingham, UK, 2Washington University, St. Louis, USA

Abstract

The tumour suppressor gene product LIMD1 is a nucleo-cytoplasmic shuttling protein implicated in the regulation of retinoblastoma protein (pRB) function and lung cancer development. To identify additional functions of LIMD1 we performed a yeast-2 hybrid screen which revealed a specific interaction with HIF-prolyl hydroxylase 1 (PHD1). In normoxia (21% oxygen) the HIF-prolyl hydroxylases (PHD1, 2 and 3) hydroxylate the master transcriptional regulator HIF-1α in an oxygen dependent manner. Proline hydroxylation of HIF1α induces binding of the von-Hippel-Lindau (pVHL) tumour suppressor, the recognition component of a multiprotein ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation by the 26S proteasome. Prevention of HIF-induced transcriptional is pivotal in repressing numerous tumourigenic processes including angiogenesis, neovascularisation and metastasis.

We report the novel findings that LIMD1 directly interacts with PHD1, 2 and 3, whilst indirectly interacting with pVHL both in vitro and in vivo. The ability of LIMD1 to interact with the PHDs and pVHL is reflected in functional assays monitoring the level of transcriptional activation from a hypoxic response element (HRE), whereby expression of LIMD1 inhibits HRE activation. shRNA mediated repression of endogenous LIMD1 also results in HRE activation. Furthermore, Limd1-/- mouse embryonic fibroblasts (MEFs) have markedly increased HRE activation following incubation in hypoxia in comparison with the wild type MEFs. In summary binding and functional studies suggest a mechanism whereby LIMD1 may modulate PHD function and HIF-1 activity. As LIMD1 is encoded within the putative tumor suppressor region 3p21.3 and is frequently down regulated in human lung tumours, the ability of LIMD1 to positively regulate PHD function may represent a novel tumour suppressive function.

Acknowledgements

This work was supported by funds from the British Lung Foundation and BBSRC (to T.V.S.).

The tumour suppressor gene product LIMD1 is a nucleo-cytoplasmic shuttling protein implicated in the regulation of retinoblastoma protein (pRB) function and lung cancer development. To identify additional functions of LIMD1 we performed a yeast-2 hybrid screen which revealed a specific interaction with HIF-prolyl hydroxylase 1 (PHD1). In normoxia (21% oxygen) the HIF-prolyl hydroxylases (PHD1, 2 and 3) hydroxylate the master transcriptional regulator HIF-1α in an oxygen dependent manner. Proline hydroxylation of HIF1α induces binding of the von-Hippel-Lindau (pVHL) tumour suppressor, the recognition component of a multiprotein ubiquitin-ligase complex, initiating HIF-1α ubiquitylation and degradation by the 26S proteasome. Prevention of HIF-induced transcriptional is pivotal in repressing numerous tumourigenic processes including angiogenesis, neovascularisation and metastasis.

We report the novel findings that LIMD1 directly interacts with PHD1, 2 and 3, whilst indirectly interacting with pVHL both in vitro and in vivo. The ability of LIMD1 to interact with the PHDs and pVHL is reflected in functional assays monitoring the level of transcriptional activation from a hypoxic response element (HRE), whereby expression of LIMD1 inhibits HRE activation. shRNA mediated repression of endogenous LIMD1 also results in HRE activation. Furthermore, Limd1-/- mouse embryonic fibroblasts (MEFs) have markedly increased HRE activation following incubation in hypoxia in comparison with the wild type MEFs. In summary binding and functional studies suggest a mechanism whereby LIMD1 may modulate PHD function and HIF-1 activity. As LIMD1 is encoded within the putative tumor suppressor region 3p21.3 and is frequently down regulated in human lung tumours, the ability of LIMD1 to positively regulate PHD function may represent a novel tumour suppressive function.

Acknowledgements

This work was supported by funds from the British Lung Foundation and BBSRC (to T.V.S.).

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