Adaptor Protein Ruk₁ Forms Protein-Protein Complexes with Endonuclease Activity in HEK293 Cells

Yu. Ya. Kit¹, V. R. Drel¹, O. I. Petriv¹, V. A. Kovalyova¹, G. Yu. Shuvaeva¹, O. Yu. Palivoda¹, E. I. Vovk¹, Ya. P. Bobak¹, J. Rzeszowska-Wolny⁴, I. T. Gout³, V. L. Buchman², and L. B. Drobot^1*

¹Institute of Cell Biology, National Academy of Sciences of Ukraine, ul. Dragomanova 14/16, Lviv 79005, Ukraine; fax: (380-322) 72-0646; E-mail: drobot@biochem.lviv.ua

²University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom; fax: (440-131) 650-6576; E-mail: v.buchman@ed.ac.uk

³Ludwig Institute for Cancer Research, Courtauld Building, 91 Riding House Street, London W1P 8BT, United Kingdom; fax: (440-20) 787-84040; E-mail: ivan@ludwig.ucl.ac.uk

⁴M. Sklodowska-Curie Memorial Institute, Branch in Gliwice, Wybrzeze AK 15, 44-100, Gliwice, Poland; fax: (48-32) 231-3512; E-mail: jwolny@io.gliwice.pl

^* To whom correspondence should be addressed.

Received August 26, 2002

---

The structural and functional organization of the adaptor protein Ruk₁ 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 Ruk₁ 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 Ruk₁ with a Glu-epitope at the C-terminus (Ruk₁ 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 Ruk₁ Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the Ruk₁ Glu-tagged preparation by retardation of electrophoretic mobility of ³²P-labeled oligodeoxyribonucleotides in gel. The Ruk₁ 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 Ruk₁ 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 Ruk₁ protein was identified in the fractions. It was concluded that the adaptor protein Ruk₁ forms complexes with endonucleases in HEK293 cells.

---

KEY WORDS: adaptor proteins, SH3-domain, Ruk₁, CIN85, SETA, protein-protein interactions, heparin-Sepharose, DNAse activity

---