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Nuclear Translocation and DNA Recognition Signals Colocalized within the bZIP Domain of Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein CREB

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School Boston, Massachusetts 02114

Address requests for reprints to: Dr. Gérard Waeber, Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114.

Abstract

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein. We conclude that 1) the NTS and DNAbinding bZIP domains of CREB are colocalized; 2) dimerization of CREB is not necessary for nuclear translocation; and 3) any proposed biological functions for the truncated CREB consisting of the transcriptional transactivation domain would probably take place in the cytoplasm.

FOOTNOTES

This work was supported in part by USPHS Grants DK-30457 and DK-25532.

Received for publication April 17, 1991. Revision received June 7, 1991. Accepted for publication June 7, 1991.

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