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 Ca 2+ dependent, while the second is not. Peptide fingerprint analysis of the purified proteins identified three putative CSN7 kinases.