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. 2021 Nov 25;10:e68473. doi: 10.7554/eLife.68473

Figure 3. Phosphorylation of Thr143 and Thr149 stabilizes the interface between CycT1 and CDK9.

Figure 3.

(A) Model of P-TEFb where CycT1 is phosphorylated at Thr143 and Thr149. The model was created by adding phosphates to Thr143 and Thr149 in the published crystal structure of P-TEFb (PDB ID 3MI9), followed by energy minimization and molecular dynamics (MD) simulations. Residues predicted to interact with Thr143 and Thr149 are Gln73 in CycT1 and Lys68 in CDK9, respectively. (B) Gln73 is targeted by phosphorylated Thr143 in CycT1. WT CycT1(280) or mutant CycT1(280)Q73A proteins and CDK9 were coexpressed in the presence of bortezomib (+/- signs on top) in 293T cells. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1 and CDK9 proteins. (C) Lys68 in CDK9 is targeted by phosphorylated Thr149 in CycT1. WT CDK9 or mutant CDK9K68A proteins and CycT1(280) were coexpressed in the presence of bortezomib (+/- signs on top) in 293T cells. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1 and CDK9 proteins. (D) Mutations of K68A in CDK9 and Q73A in CycT1 attenuate cooperatively the binding between CycT1 and CDK9, equivalently to the mutant CycT1TT143149AA protein. WT CycT1(280) or mutant CycT1(280)Q73A proteins and WT CDK9 or mutant CDK9K68A proteins were coexpressed in the presence of bortezomib (+/- signs on top) in 293T cells. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1 and CDK9 proteins.