NM23-H1 and NM23-H2 Repress Transcriptional Activities of Nuclease-hypersensitive Elements in the Platelet-derived Growth Factor-A Promoter

Abstract

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3-portions of both 5-SHS strands in either singlestranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5-and 3-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (؊82 to ؉8). Activity of the negative regulatory region (؊1853 to ؊883), which contains the 5-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.The platelet-derived growth factor (PDGF) 1 family consists of three structurally similar glycoproteins (M r 30,000) that induce proliferation and other growth-related effects in cells of mesenchymal origin. These proteins arise from covalent dimerization of two PDGF subunits, designated the A-chain and B-chain, yielding the heterodimer PDGF-AB and two homodimers, PDGF-AA and PDGF-BB (1, 2). PDGF was implicated in tumorigenesis following the discovery of high sequence homology between the PDGF B-chain (PDGF-B) and the viral oncogene, v-sis (for a review, see Ref.3). Other studies suggest that both PDGF-A and PDGF-B may also mediate tumor progression to the metastatic phenotype (4, 5). Transcription of the PDGF-A gene is regulated by several enhancer and silencer elements that are poly-purine/pyrimidine-rich and possess a high degree of single-stranded, non-B DNA structure. Other laboratories (6, 7) as well as our own (8) have demonstrated that a highly GC-rich and nuclease-hypersensitive element (PDGF-A NHE) in the proximal 5Ј-flanking sequence of the PDGF-A promoter (Ϫ82 to Ϫ40) contributes most of the basal transcriptional activity of the gene. This activity is mediated by the binding of members of the Sp1 family of transcription factors and can be induced in vascular endothelial cells by phorbol ester treatment through displacement of Sp1 and Sp3 by the early growth response factor Egr-1 (7, 9) or repressed by binding of the Wilms' tumor gene product WT1 (10). More recently, we local...

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

“…In addition to the phosphoryl transfer reactions, they can catalyze various types of DNA cleavage (9 -12) and activate transcription (13)(14)(15). The involvement of NM23 in DNA repair was initially proposed on the basis of a covalent, lysine-mediated mechanism by which NM23-H2/NDK-B binds and cleaves DNA (9), a mechanism known as the signature of base excision repair lyases (16).…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…These multiple functions assigned to the NM23 family of proteins have thus far been difficult to reconcile because the exact cellular function of NM23 has remained unclear. However, beyond nucleoside diphosphate kinase activity several other molecular activities have been linked to NDPKs, such as histidine-dependent protein kinase (histidine phosphotransferase) [18,19] and nuclease activity [20,21], as well as lipid bilayer binding [22][23][24][25][26].…”

NDP Kinase 7 Is a Conserved Microtubule-Binding Protein Preferentially Expressed in Ciliated Cells

“…None of them contains any other known functional domains. Group I enzymes are expressed ubiquitously (Lacombe et al, 2000) and involved in a wide variety of cellular processes that include suppression of tumor metastasis (Steeg et al, 1988;Chang et al, 1994;Leone et al, 1991;MacDonald et al, 1996), transcription regulation (Postel et al, 2000), DNA cleavage (Postel et al, 2000;Ma et al, 2002;Ma et al, 2004;Yoon et al, 2005) and p53-induced apoptosis (Tsuiki et al, 1999). Some NDP kinases have been abundantly found in specific locations of postmeiotic male germ cells and in mature sperm flagella, suggesting that they are involved in spermiogenesis or sperm motility (Munier et al, 2003).…”