NM23-H1 Tumor Suppressor Physically Interacts with Serine-Threonine Kinase Receptor-associated Protein, a Transforming Growth Factor-β (TGF-β) Receptor-interacting Protein, and Negatively Regulates TGF-β Signaling

Hyun-A Seong,Haiyoung Jung,Hyunjung Ha

doi:10.1074/jbc.m609832200

Abstract

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.

Highlights

In humans, the eight NM23 genes that have been identified to date, NM23-H1-specific siRNA(b) (NM23-H1), NM23-H2, NM23-H3, NM23-H4, NM23-H5, NM23-H6, NM23-H7, and NM23-H8, encode NDP kinases or homologous isoforms (2)
serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-␤ receptor-interacting protein (23). Based on this previous result, we investigated whether NM23-H1 interacted with STRAP in mammalian cells by performing a set of cotransfection experiments using
These data corroborated the results of the yeast two-hybrid screen and demonstrated that NM23-H1 physically interacts with STRAP in cells

Summary

Introduction

The eight NM23 genes that have been identified to date, NM23-H1, NM23-H2, NM23-H3, NM23-H4, NM23-H5, NM23-H6, NM23-H7, and NM23-H8, encode NDP kinases or homologous isoforms (2). NM23 family proteins have been shown to associate with several cellular proteins, including glyceraldehyde-3-phosphate dehydrogenase (9), Hsc (70-kDa heat shock cognate protein) (10), telomere (11), ROR␣ (retinoid acid receptor-related orphan receptor ␣)/RZR␤ (retinoid Z receptor ␤) (12), Rad, a Ras-related small GTPase (13), creatine kinase and antioxidant protein (14), and thromboxane A2 receptor, a G protein-coupled receptor (6). These results suggest that the identification of additional binding partners of NM23 proteins will provide greater insight into the regulation and biological function of NM23 family proteins. We report that the physical association of NM23-H1 with STRAP in vivo is important for the negative regulation of TGF-␤-mediated signaling as well as NM23-H1 tumor suppressor activity

Methods

Results

Conclusion