Andreas Krehan

Multiple forms of protein kinase CK2 present in leukemic cells: in vitro study of its origin by proteolysis.

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2α′ was more resistant to these proteases than CK2α. When these proteases were assayed on the reconstituted (α2β2 holoenzyme, a 37 kDaα-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2α deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2α. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells. (Mol Cell Biochem 191: 229–234, 1999)

Intermolecular contact sites in protein kinase CK2

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55–70 of subunit β (β55–70) and 65–80 of subunit α (α65–80). This has been supported by crosslinking of subunits with peptides α65–80 and β55–70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-α65–80; anti-β55–70). Functionally, β55–70 is a negative regulatory region for the kinase activity of subunit α. The opposite, stimulatory property of subunit β has been assigned to its C-terminal part. Subdivision of peptide β 155–181, that has stimulatory effect, into overlapping peptides and assaying for α binding and binding competition revealed a tight physical contact at β162–175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit α as indicated by crosslinking and peptide competition. The crosslinking data also confirm a β-β contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment. (Mol Cell Biochem 191: 21–28, 1999)