Expression of DNA‐Dependent Protein Kinase Holoenzyme Upon Induction of Lymphocyte Differentiation and V(D)J Recombination

Abstract

Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (1L-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-PK,) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PK,, 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-PK,, 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.

The DNA-dependent protein kinase

“…Furthermore, elevated levels or activity of Ku have been reported in response to cellular growth state (Cai et al 1994) possibly reflecting the increased need for efficient DNA DSB repair in rapidly dividing cells. DNA-PK activity is found to be induced to some extent when lymphoid cells are induced to undergo site-specific V(D)J recombination or switch recombination (Grawunder et al 1996); two processes that rely on DNA-PK function (e.g., see Rolink et al 1996;Casellas et al 1998;Manis et al 1998). However, as this result was obtained from cells in culture, it remains unclear if this activation of DNA-PK occurs in the whole animal.…”

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…Recently, we have con®rmed that Ku is localized in the nucleus as a heterodimer of Ku70 and Ku80 subunits in interphase cells (Koike et al, 1998). On the other hand, accumulating evidence suggests that Ku is not only localized in the nuclei but also in the cytoplasm (Fewell and Ku , 1996;Grawunder et al, 1996). Thus, we examined the cellular localization of the Ku70 and Ku80 proteins in HeLa-S3 cells using confocal laser scanning microscopy ( Figure 1A).…”

“…On the other hand, although Ku has been thought to be localized and functioned only in the nucleus, several studies have reported cytoplasmic or cell surface localization of Ku proteins in various cell types (Prabhakar et al, 1990;Danziel et al, 1992;Reeves, 1992;Grawunder et al, 1996). Furthermore, changes in the subcellular localization of Ku could be controlled by a variety of external growth-regulating stimuli (Le Romancer et al, 1994).…”

The catalytic subunit of DNA–protein kinase (DNA-PKcs) is not required for Ig class-switch recombination

“…Interestingly, CSR has been shown to occur in DNA-PKcs-deficient B cells in Ig transgenic SCID mice (11,12). SCID cells express a truncated and enzymatically dead DNA-PKcs protein (13)(14)(15)(16). Thus, the SCID results could be taken to imply that DNA-PKcs plays only a structural role in CSR.…”