The helix-loop-helix proteins (salivary-specific cAMP response element-binding proteins) can modulate cAMP-inducible RP4 gene expression in salivary cells.
Salivary-specific and cAMP-inducible expression of the rat proline-rich protein gene RP4 is dependent on a 28-base pair sequence of a salivary-specific cAMP response element (SCRE) (Lin, H. H., and Ann, D. K. (1992) Gene Expression 2, 365-377). To unravel its trans-acting factor(s), we used double-stranded oligoprobes corresponding to the SCRE to screen a randomly primed lambda gt11 cDNA expression library made from RNA of rat salivary cells. In this report, we describe the cDNA cloning of these helix-loop-helix SCRE-binding proteins (SCBPs) and demonstrate that there are at least three isoforms in salivary cells, namely SCBP alpha, SCBP beta, and SCBP gamma. RNA polymerase chain reaction and sequence analyses further confirmed the existence of these three different SCBP isoforms, which code for putative proteins of 707, 706, and 682 amino acids, respectively. Expression of the cloned SCBP cDNAs in salivary cells stimulates the expression of a cotransfected reporter construct containing multicopies of the SCRE cloned upstream of the thymidine kinase promoter and the chloramphenicol acetyltransferase structural gene. This stimulation is much more pronounced in transfections in which SCBP alpha and SCBP beta are cotransfected than when they are transfected individually. Furthermore, when low concentrations of SCBP alpha and SCBP beta are cotransfected with the SCRE reporter gene, coexpression of the catalytic subunit of protein kinase A is required to efficiently activate the expression of the reporter gene. These results strongly suggest that the observed stimulation of the SCRE is achieved through the coordinated expression of the SCBP alpha, SCBP beta, and protein kinase A activities, perhaps via a direct association of the two SCBPs and their phosphorylation by protein kinase A. We conclude that the isolated SCBP alpha and SCBP beta cDNAs encode transcription activators that participate in the control of the inducible RP4 gene expression in salivary cells.
Lin
H H
HH
Department of Pharmacology Medical School, University of Minnesota, Minneapolis 55455.
Li
W Y
WY
Ann
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D055232:Proline-Rich Protein Domains; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012469:Salivary Glands / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D015500:Chloramphenicol O-Acetyltransferase; D000242:Cyclic AMP; D004247:DNA; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D010455:Peptides; D012313:RNA; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D014157:Transcription Factors; C081068:Tcf12 protein, rat