Regulation of elongation factor-1alpha expression by growth factors and anti-receptor blocking antibodies.

The epidermal growth factor (EGF) family and its receptors regulate normal and cancerous epithelial cell proliferation, a process that could be suppressed by anti-receptor blocking antibodies. Polypeptide elongation factor-1alpha (EF-1alpha) is a multifunctional protein whose levels are positively correlated with the proliferative state of cells. To identify genes, whose expression may be modulated by anti-receptor blocking antibodies, we performed a differential display screening and isolated differentially expressed cDNAs. Isolates from one clone were 100% identical to human EF-1alpha. Both EGF and heregulin-beta1 (HRG) induced EF-1alpha promoter activity and mRNA and protein expression. Growth factor-mediated EF-1alpha expression was effectively blocked by pretreatment with humanized anti-EGF receptor antibody C225 or anti-human epidermal growth factor receptor-2 (HER2) antibody herceptin. Mutants and pharmacological inhibitors of p38(MAPK) and MEK, but not phosphatidylinositol 3-kinase, suppressed both constitutive and HRG-induced stimulation of EF-1alpha promoter activity in MCF-7 cells. Deletion analysis of the promoter suggested the requirement of the -393 to -204 region for growth factor-mediated transcription of EF-1alpha. Fine mapping and point mutation studies revealed a role of the SP1 site in the observed HRG-mediated regulation of the EF-1alpha promoter. In addition, we also provide new evidence to suggest that HRG stimulation of the EF-1alpha promoter involves increased physical interactions with acetylated histone H3 and histone H4. These results suggest that regulation of EF-1alpha expression by extracellular signals that function through human EGF receptor family members that are widely deregulated in human cancers and that growth factor regulation of EF-1alpha expression involve histone acetylation.

The human elongation factor 1 A-2 gene (EEF1A2): complete sequence and characterization of gene structure and promoter activity.

The eukaryotic elongation factor 1 A (eEF1A, formerly EF1alpha) is a key factor in protein synthesis, where it promotes the transfer of aminoacylated tRNAs to the A site of the ribosome. Two differentially expressed isoforms of eEF1A, designated eEF1A-1 and eEF1A-2, are found in mammals. Here we report the isolation and sequencing of the gene (HGMW-approved symbol EEF1A2) coding for the human eEF1A-2 isoform. Furthermore, we characterize the gene structure and the activity of the promoter. Isolation of overlapping clones from human libraries revealed that the human eEF1A-2 gene spans approximately 10 kb and consists of eight exons. The intron-exon boundaries of human EEF1A2 and EEF1A1 are conserved, yet the gene of the eEF1A-2 isoform is larger than the eEF1A-1 gene because of enlarged introns. Primer extension analysis identified the predominant transcription start site 166 bp upstream of the AUG codon. The start site maps to an adenine located within a consensus initiator element. Sequencing of a 2-kb 5'-flanking promoter region revealed no TATA element. However, several putative cis-regulatory elements were discovered. The 5'-promoter activity was characterized by transient transfection experiments. Progressive deletions of the upstream promoter region defined a minimal promoter region, ranging from -16 to +92, that is sufficient to drive transcription.

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.

Different Sp1 family members differentially affect transcription from the human elongation factor 1 A-1 gene promoter.

The GC box is an important transcriptional regulatory element present in the promoters of many mammalian genes. In the present study we examine the effect of known GC-box-binding proteins on the promoter of the human elongation factor 1 A-1 (hEF1A-1) gene in human HeLa cells and Drosophila SL2 cells. In HeLa cells co-transfection with the GC-box-binding protein BTEB resulted in a 4-10-fold increase in hEF1A-1 promoter activity. This stimulation was dependent on a single GC box located between positions -69 and -50 of the promoter. Little or no effect was observed of other GC-box-binding proteins including Sp1, Sp3, Sp4 and BTEB2. In SL2 cells stimulation by Sp1 and Sp3 through the single GC box of the proximal promoter led to 13-fold and 21-fold increases respectively in promoter activity. Inclusion of further upstream sequences resulted in high levels of expression when Sp1 or Sp3 was co-transfected with the reporter plasmid. In this setting Sp1 stimulated transcription by 750-fold, whereas Sp3 was even more potent, yielding a 1150-fold stimulation. Mobility-shift assays performed with the promoter-proximal GC box demonstrated the binding of Sp1, Sp3 and Sp4 to this sequence. To our knowledge, the present study represents the first comparison of all known GC-box-binding proteins on a natural promoter.