Fig. 1: In vitro reconstitution of a minimal system recapitulating T-loop phosphorylation of Plk1 by Bora and AURKA.
a Domain architecture of Homo sapiens AURKA, Bora, and Plk1. The positions of the T-loop phospho-regulatory sites of AURKA (T288) and Plk1 (T210) are indicated. Cy motifs in Bora correspond to Cyclin-binding motifs that mediate substrate targeting. b Two-step in vitro reconstitution of T-loop phosphorylation on T210 of Plk1 (pT210) by AURKA and Bora1–224. In step 1, Bora is phosphorylated by CyclinA2-Cdk2 kinase (yielding pBora). In step 2, phosphorylated Bora from the reaction mix in step 1 is incubated with AURKA and the catalytically dead mutant Plk1K82R in full-length (FL), polo-box domain deleted (∆PBD), or kinase domain-only (KD) forms. During step 2, AURKA phosphorylates Plk1 (noted pT210 Plk1K82R) and Bora or pBora (noted ppBora). c Western blot analysis of kinase reactions carried out with Bora1–224 phosphorylated (+) or not (−) by CyclinA2-Cdk2 (step 1) in the presence of Plk1K82R (FL or ∆PBD or KD) and AURKA (step 2). Schematics of the Plk1 constructs used as substrates are shown at bottom. Blots were probed with antibodies to Bora, AURKA, and phosphoT210 Plk1 or pan Plk1 as indicated (from top to bottom). Note that pBora phosphorylated by AURKA displays slower migration on SDS-PAGE compared to pBora phosphorylated only by CyclinA2-Cdk2. In the blot performed with the anti-pT210 Plk1 antibody, the asterisk denotes cross reactivity with the pT288 residue of AURKA.
