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

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)-β-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 Cys145 of NM23-H1 and Cys 152 and Cys270 of STRAP but did not appear to involve Cys4 and Cys109 of NM23-H1, suggesting that a disulfide linkage involving Cys145 of NM23-H1 and Cys152 or Cys 270 of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or β-mercaptoethanol but not H2O2. Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-β signaling in a dose-dependent manner, enhanced stable association between the TGF-β receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-β-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-β 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-β-dependent biological activity as well as NM23-H1 activity. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.