Adaptor Protein Ruk sub(1) Forms Protein-Protein Complexes with Endonuclease Activity in HEK293 Cells

The structural and functional organization of the adaptor protein Ruk sub(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 sub(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 sub(1) with a Glu-epitope at the C-terminus (Ruk sub(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 sub(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 sub(1) Glu-tagged preparation by retardation of electrophoretic mobility of super(32)P-labeled oligodeoxyribonucleotides in gel. The Ruk sub(1) Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl sub(2) and heparin inhibited the DNAse activity. These findings suggest the presence of DNases associated with the Ruk sub(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 ZnCl sub(2) 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 sub(1) protein was identified in the fractions. It was concluded that the adaptor protein Ruk sub(1) forms complexes with endonucleases in HEK293 cells.