Adaptor protein Ruk1 forms protein-protein complexes with endonuclease activity in HEK293 cells.
The structural and functional organization of the adaptor protein Ruk(1) is characterized by the presence of three SH3-domains at the N-terminus followed by Pro- and Ser-rich sequences and a C-terminal coiled-coil region. Multiple modules in the Ruk(1) structure involved in protein-protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein Ruk(1) with a Glu-epitope at the C-terminus (Ruk(1) Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to the 85-kD Ruk(1) Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk(1) Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The Ruk(1) Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk(1) protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with DNase activity and containing proteins with molecular weights of 83, 80, and 72 kD were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83-kD protein immunologically related to the Ruk(1) protein was identified in the fractions. It was concluded that the adaptor protein Ruk(1) forms complexes with endonucleases in HEK293 cells.
Kit
Yu Ya
YY
Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 79005, Ukraine.
Drel
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Petriv
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Kovalyova
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Shuvaeva
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Palivoda
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Vovk
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Bobak
Ya P
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Rzeszowska-Wolny
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Gout
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Buchman
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Drobot
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D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C433810:SH3KBP1 protein, human; D004247:DNA; D003851:Deoxyribonucleases