Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene.

Myc-binding protein-1 (MBP-1) is a 37-kDa protein with sequence homology to the 3' portion of the alpha-enolase gene. alpha-Enolase is a 48-kDa protein, which plays a critical role in the glycolytic pathway. MBP-1 binds to the c-myc P2 promoter and down-regulates c-myc expression. We have investigated the role of alpha-enolase in regulation of the c-myc protooncogene. RNase protection assay shows that alpha-enolase is transcribed into a single RNA species in HeLa cells. A start codon, 400 base pairs downstream of the alpha-enolase ATG, corresponds to the MBP-1 ATG, suggesting that MBP-1 is an alternative translation initiation product of the alpha-enolase RNA. Domain mapping was performed using constructs containing truncations of the alpha-enolase gene. In vitro binding to the c-myc gene was abolished after deletion of the N-terminal portion of alpha-enolase. In order to determine the relationship between DNA binding activity and transcription inhibition, we performed co-transfection assays in HeLa cells. These studies confirmed that an N-terminal deletion of alpha-enolase is unable to down-regulate c-myc promoter activity. Our data suggest that alpha-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product MBP-1, which is distinct from its role as a glycolytic enzyme.

[1]  U. Schibler,et al.  CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Wold,et al.  Binding properties of replication protein A from human and yeast cells , 1992, Molecular and cellular biology.

[3]  G. Bornkamm,et al.  Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene , 1987, Molecular and cellular biology.

[4]  E. Dickson,et al.  Endothelial cell hypoxia associated proteins are cell and stress specific , 1993, Journal of cellular physiology.

[5]  Hans R. Schöler,et al.  New type of POU domain in germ line-specific protein Oct-4 , 1990, Nature.

[6]  M. Hendrix,et al.  Human breast carcinoma cells transfected with the gene encoding a c-myc promoter-binding protein (MBP-1) inhibits tumors in nude mice. , 1995, Cancer research.

[7]  N. Hay,et al.  Regulatory elements that modulate expression of human c-myc. , 1987, Genes & development.

[8]  F. Bosch,et al.  Evidence from transgenic mice that myc regulates hepatic glycolysis , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  W. Schaffner,et al.  cDNA cloning of human N-Oct3, a nervous-system specific POU domain transcription factor binding to the octamer DNA motif. , 1993, Nucleic acids research.

[10]  A. Patel,et al.  myc function and regulation. , 1992, Annual review of biochemistry.

[11]  K. Sakimura,et al.  Molecular cloning and the nudeotide sequence of cDNA to mRNA for Don-nearonal enolase (αα enolase) of rat brain and liver , 1985 .

[12]  G. Sonenshein,et al.  Transcriptional and posttranscriptional control of c-myc gene expression in WEHI 231 cells , 1986, Molecular and cellular biology.

[13]  S. Hedges,et al.  Molecular evidence for the origin of birds. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Sassone-Corsi,et al.  Alternative usage of initiation codons in mRNA encoding the cAMP-responsive-element modulator generates regulators with opposite functions. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[15]  W. D. de Jong,et al.  Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein , 1988, The Journal of cell biology.

[16]  J. Lélias,et al.  Nucleoticle sequences of cDNAs α and γ enolase mRNAs from mouse brain , 1990 .

[17]  Jonathan A. Cooper,et al.  Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus , 1983, Nature.

[18]  C. Croce,et al.  Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Leder,et al.  Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in burkitt lymphoma , 1984, Cell.

[20]  H. Farber,et al.  Non-neuronal Enolase Is an Endothelial Hypoxic Stress Protein (*) , 1995, The Journal of Biological Chemistry.

[21]  R. Ray,et al.  Cloning and characterization of a human c-myc promoter-binding protein , 1991, Molecular and cellular biology.

[22]  N. Segil,et al.  Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence. , 1988, The Biochemical journal.

[23]  G. Barbieri,et al.  Structure of the human gene for α‐enolase , 1990 .

[24]  S. Cogoi,et al.  Alternative translation initiation site usage results in two functionally distinct forms of the GATA-1 transcription factor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Ray,et al.  Inhibition of human immunodeficiency virus type 1 replication by a cellular transcriptional factor MBP‐1 , 1997, Journal of cellular biochemistry.

[26]  D. Chaudhary,et al.  The c-myc promoter binding protein (MBP-1) and TBP bind simultaneously in the minor groove of the c-myc P2 promoter. , 1995, Biochemistry.

[27]  P. Leder,et al.  Trans-acting elements modulate expression of the human c-myc gene in Burkitt lymphoma cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Mark Groudine,et al.  A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells , 1986, Nature.

[29]  M. Groudine,et al.  Control of c-myc regulation in normal and neoplastic cells. , 1991, Advances in cancer research.

[30]  V. Fischetti,et al.  α-Enolase, a Novel Strong Plasmin(ogen) Binding Protein on the Surface of Pathogenic Streptococci* , 1998, The Journal of Biological Chemistry.

[31]  A. Prats,et al.  Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells , 1996, The Journal of cell biology.

[32]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[33]  S. Cory,et al.  Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumours. , 1983, The EMBO journal.

[34]  R. Sears,et al.  The alternatively initiated c-Myc proteins differentially regulate transcription through a noncanonical DNA-binding site. , 1994, Genes & development.

[35]  H. Towle.  Metabolic Regulation of Gene Transcription in Mammals (*) , 1995, The Journal of Biological Chemistry.

[36]  F. Bosch,et al.  Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Jordi Félez,et al.  Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor. , 1991, Biochemistry.

[38]  O. MacDougald,et al.  A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Ray,et al.  Functional Domains of c-myc Promoter Binding Protein 1 Involved in Transcriptional Repression and Cell Growth Regulation , 1999, Molecular and Cellular Biology.

[40]  M. Cole.  The myc oncogene: its role in transformation and differentiation. , 1986, Annual review of genetics.