NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling.
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.
Seong
Hyun-A
HA
Department of Biochemistry, Research Center for Bioresource and Health, Biotechnology Research Institute, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Jung
Haiyoung
H
Ha
Hyunjung
H
D021581:Active Transport, Cell Nucleus; D017209:Apoptosis; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D003545:Cysteine / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases; D009363:Neoplasm Proteins / Q000737:chemistry*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000378:metabolism
D054778:NM23 Nucleoside Diphosphate Kinases; D009363:Neoplasm Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C403447:STRAP protein, human; D016212:Transforming Growth Factor beta; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D003545:Cysteine