Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2’s anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.