Characterization and purification of kinase activities against Arabidopsis COP9 signalosome subunit 7

The COP9 signalosome (CSN) is a multisubunit protein complex that intersects the ubiquitin (Ub)-proteasome pathway at several junctions. The CSN associates with several protein kinases that target key regulatory proteins that themselves are targets for Ub-dependent degradation, and several subunits themselves are phosphoproteins. We previously reported that Arabidopsis CSN7 is a phosphoprotein. To identify the kinases responsible for CSN7 phosphorylation, we biochemically purified CSN7-kinsae activities from cauliflower through a four-step purification procedure that resulted in a ca. 300-fold enrichment. One of these activities is Ca²⁺ dependent, while the second is not. Peptide fingerprint analysis of the purified proteins identified three putative CSN7 kinases.