Figure 5
From: Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
Use of Arabidopsis CDKF;1 as a CAK. (a) StrepIII-tagged CDKA;1 was co-expressed in bacteria with either GST-tagged Cak1 (from S. cerevisiae), or GST-tagged WEE1 (Arabidopsis), or GST-tagged CDKF;1 (Arabidopsis). The phosphorylation status of CDKA;1 was determined by western blots using antibodies against Phospho-Tyr (pTyr15), typically catalyzed by the kinase WEE1, and Phospho-Thr161 in the T-loop of CDKA;1 (pThr161), typically catalyzed by the activating kinase, either Cak1 or CDKF;1. Arabidopsis CDKF;1 can efficiently phosphorylate the T-loop of CDKA;1 at the residue T161 but not Y15 in the P-loop. In contrast, the kinase WEE1 can phosphorylate CDKA;1 at residue Y15 but not T161. (b) Complexes binding to a Ni-NTA column (His-tagged proteins) were purified from bacteria either expressing HisMBP-tagged SDS together with CDKF;1 as a CAK, or HisMBP-SDS together with StrepIII-tagged CDKA;1, or HisMBP-SDS together with CDKF;1 and StrepIII-tagged CDKA;1. High levels of kinase activity could be recovered from CDKA;1-SDS complexes that were activated by the endogenous Arabidopsis CAK, CDKF;1.