Metastasis suppressor function of NM23-H1 requires its 3'-5' exonuclease activity.
The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3'-5' exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3'-5' exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3'-5' exonuclease alone (Glu(5) to Ala, or E(5) A), the NDPK and histidine kinase activities tandemly (Y(52) A, H(118) F) or all 3 activities simultaneously (K(12) Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E(5) A and K(12) Q. The H(118) F mutant, which lacked both the NDPK and histidine kinase while retaining the 3'-5' exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3'-5' exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.
Zhang
Qingbei
Q
Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.
McCorkle
Joseph R
JR
Novak
Marian
M
Yang
Mengmeng
M
Kaetzel
David M
DM
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000556:secondary; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous
D054778:NM23 Nucleoside Diphosphate Kinases; D018698:Glutamic Acid; D006639:Histidine; D011494:Protein Kinases; D000071677:Histidine Kinase; D005092:Exonucleases; D008239:Lysine