Identification and Functional Analysis of Novel cAMP Response Element Binding Protein Splice Variants Lacking the Basic/Leucine Zipper Domain

Abstract

Two novel cAMP response element binding protein (CREB) splice variants were found by reverse transcription-polymerase chain reaction cloning by using mouse brain RNA as a template. One splice variant, named Δ-14, lacks 14 nucleotides at the beginning of exon 9 of the CREBΔ isoform. The other, named Δ-35, lacks 35 nucleotides at the beginning of exon 8 of CREBΔ. These nucleotide deletions cause frame shifts for codon usage, producing proteins which conserve the major phosphorylation site (Ser¹³³) but lack the basic/leucine zipper domain, which is essential for binding to DNA and to other transcription factors. Both variants are widely expressed in peripheral tissues, but are enriched in brain, thymus, and testis. CREBΔ-14 and Δ-35 variant proteins were expressed by using an in vitro translation system and by transfecting into human embryonic kidney 293 cells. Both variants were detected by a CREB antibody that recognizes the CREBΔ amino terminus, but not by an antibody which recognizes the CREBΔ carboxy terminus, as would be predicted based on the frame shift. Activation of the cAMP pathway increased phospho-CREB immunoreactivity, indicating that these variants are substrates of cAMP-dependent protein kinase. In addition, immunocytochemical analysis demonstrated that CREBΔ-14 and Δ-35 are primarily cytosolic, whereas CREBα is predominantly in the nucleus. Finally, expression of CREBΔ-14 or Δ-35 decreased cAMP responsive element-chloramphenicol acetyltransferase reporter activity, demonstrating that both can function as repressors of endogenous CREB.