Phosducin interacts with the G-protein betagamma-dimer of ciliate protozoan Blepharisma japonicum upon illumination.
Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the betagamma-subunits of G-protein (Gbetagamma) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gbetagamma. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gbetagamma binding by this molecule. Possible formation of the Pdc-Gbetagamma complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gbetagamma and generation of the Pdc-Gbetagamma complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc-Gbetagamma complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gbeta. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.
Sobierajska
Katarzyna
K
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, PL-02-093 Warsaw, Poland.
Fabczak
Hanna
H
Fabczak
Stanisław
S
D048948:14-3-3 Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D016798:Ciliophora / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D003593:Cytoplasm / Q000378:metabolism / Q000528:radiation effects; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004735:Energy Transfer / Q000528:radiation effects; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020555:GTP-Binding Protein Regulators / Q000737:chemistry / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D047468:Immunoprecipitation; D008027:Light; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D021381:Protein Transport / Q000528:radiation effects
D048948:14-3-3 Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010750:Phosphoproteins; C065631:phosducin