Adaptor protein RuK1forms protein-protein complexes with endonuclease activity in HEK293 cells

Yu Ya Kit, V. R. Drel', O. I. Petrov, V. A. Kovaleva, G. Yu Shuvaeva, O. Yu Palivoda, E. I. Vovk, Ya P. Bobak, J. Rzeszowska-Wolny, I. T. Gout, V. L. Buchman, L. B. Drobot

Research output: Contribution to journalArticlepeer-review

Abstract

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. 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.

Original languageEnglish (US)
Pages (from-to)985-991
Number of pages7
JournalBiokhimiya
Volume68
Issue number7
StatePublished - 2003
Externally publishedYes

Keywords

  • Adaptor proteins
  • CIN85
  • DNAse activity
  • Heparin-Sepharose
  • Protein-protein interactions
  • Ruk
  • SETA
  • SH3-domain

ASJC Scopus subject areas

  • Chemistry(all)

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