Identification and functional analysis of novel cAMP response element binding protein splice variants lacking the basic/leucine zipper domain

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 Delta-14, lacks 14 nucleotides at the beginning of exon 9 of the CREB Delta isoform. The othe r, named Delta-35, lacks 35 nucleotides at the beginning of exon 8 of CREB Delta. These nucleotide deletions cause frame shifts for codon usage, produ cing proteins which conserve the major phosphorylation site (Ser(133)) 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. CREB Del ta-14 and Delta-35 variant proteins were expressed by using an in vitro tra nslation system and by transfecting into human embryonic kidney 293 cells. Both variants were detected by a CREB antibody that recognizes the CREB Del ta amino terminus, but not by an antibody which recognizes the CREB Delta c arboxy terminus, as would be predicted based on the frame shift. Activation of the cAMP pathway increased phospho-CREB immunoreactivity, indicating th at these variants are substrates of cAMP-dependent protein kinase. In addit ion, immunocytochemical analysis demonstrated that CREB Delta-14 and Delta- 35 are primarily cytosolic, whereas CREB alpha is predominantly in the nucl eus. Finally, expression of CREB Delta-14 or Delta-35 decreased cAMP respon sive element-chloramphenicol acetyltransferase reporter activity, demonstra ting that both can function as repressors of endogenous CREB.