Phosphorylation modulates the function of the vasoactive intestinal polypeptide receptor transcriptional repressor protein.
The transcriptional repressor for rat vasoactive intestinal polypeptide receptor 1 (VIPR-RP) is a recently isolated transcription factor. In this study, we have characterized the functional domains of VIPR-RP and the importance of phosphorylation on VIPR-RP function. Using various regions of VIPR-RP in gel mobility shift assays, we show that the amino acid sequences between positions 367 and 475 play an essential role for VIPR-RP DNA binding. Transient transfection of fusion constructs containing GAL4 DNA binding domain and different parts of VIPR-RP indicated that there are two separate transcriptional repression domains in VIPR-RP, located between amino acids 50 and 101 and between 469 and 527. We demonstrated that VIPR-RP is phosphorylated in vitro by casein kinase II on Ser-69/71 and Thr-110, and by cAMP-dependent kinase on Ser-245/361. Furthermore, by site-directed mutagenesis, we show that phosphorylation of the casein kinase II sites potentiates VIPR-RP transcriptional repression activity by enhancing its nuclear translocation, and that phosphorylation by cAMP-dependent kinase inhibits VIPR- RP transcriptional repression function without affecting its subcellular localization. These observations suggest that phosphorylation plays an important role in regulating VIPR-RP function.
Pei
L
L
Division of Endocrinology and Metabolism, Cedars-Sinai Research Institute-UCLA School of Medicine, Los Angeles, California 90048, USA. pei@cshs.org
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D047390:Casein Kinase II; D002460:Cell Line; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051818:Replication Protein C; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013912:Threonine; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C113967:Rfc1 protein, rat; D014157:Transcription Factors; D013912:Threonine; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051818:Replication Protein C