Expression of DNA-dependent protein kinase holoenzyme upon induction of lymphocyte differentiation and V(D)J recombination

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

Abstract

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL-7), and can be induced to differentiate to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gene rearrangements occur. DNA-dependent protein kinase (DNA-PK) is required for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2-/- mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that crossreacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2-/- cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J-recombination-active, early-preB and CD4-/CD8- thymocyte cell populations. Upon differentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activity, which is not detectable in SCID cells, increases in wild-type and Rag-2-/- cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.

Details

Original languageEnglish
Pages (from-to)931-40
Number of pages10
JournalEuropean Journal of Biochemistry
Volume241
Issue number3
Publication statusPublished - 1 Nov 1996
Peer-reviewedYes

External IDs

Scopus 0029916062

Keywords

Keywords

  • Animals, Antigens, Nuclear, B-Lymphocytes/enzymology, Cell Compartmentation, Cell Differentiation, Coenzymes/biosynthesis, DNA Helicases, DNA-Activated Protein Kinase, DNA-Binding Proteins/biosynthesis, Fluorescent Antibody Technique, Gene Rearrangement, B-Lymphocyte, Hematopoietic Stem Cells/enzymology, Homeodomain Proteins, Ku Autoantigen, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, SCID, Mice, Transgenic, Nuclear Proteins/biosynthesis, Protein Biosynthesis, Protein Serine-Threonine Kinases/biosynthesis, Recombination, Genetic