The carboxyl terminal domain of phosducin functions as a transcriptional activator.
In previous work, we identified a set of phosducin (Phd) isoforms with unknown function including the phosducin (Phd)-like orphan protein 1 (PhLOP1), an amino terminal truncated isoform of the retinal Phd lacking the Gbetagamma binding domain. To investigate the potential biological function of PhLOP1, PhLOP1 was fused at its amino terminus with the DNA binding domain (BD) of the yeast transcriptional factor, GAL4, and used as bait in a yeast two-hybrid screen. Two potential functional protein partners were identified during the screen: SUG1, a subunit of the 26S proteasome and a putative transcriptional mediator, and CRX, a retina- and pineal-specific transcription factor. Upon localizing the interacting domain of PhLOP1 with one of the new partners, SUG1, we found that a domain of 40 amino acids at the carboxyl terminus of Phd and PhLOP1 had intrinsic transcriptional activation activity in yeast. The transactivation activity was further confirmed in mammalian cells. This region contains an acidic domain that has been shown to be involved in the function of several transcriptional activators. In addition, we showed that Phd is cytoplasmic while PhLOP1 is localized predominantly to the nucleus when fused to an enhanced green fluorescent protein (EGFP) and transiently expressed in transfected cells, suggesting that PhLOP1 may play a distinct functional role in transcriptional regulation independent of the known Phd interaction/regulation of Gbetagamma transduction.
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Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Department of Cell and Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90089-9112, USA.
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D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C092282:PSMC5 protein, human; D010750:Phosphoproteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C106843:SUG1 protein, mammalian; D015534:Trans-Activators; D014157:Transcription Factors; C109288:cone rod homeobox protein; C065631:phosducin; D015500:Chloramphenicol O-Acetyltransferase; D046988:Proteasome Endopeptidase Complex; D000074183:ATPases Associated with Diverse Cellular Activities