Anshabo et al.
CDK9 in Cancer
FIGURE 1 | Timeline for the discovery of P-TEFb and its biological roles.
conserved sub-domains that fold into a bilobal tertiary structure help in anchoring the adenine moiety (22). The a and b non-
with smaller N-terminal and larger C-terminal lobes. The N- transferable phosphates of ATP are held in position through ionic
terminal region contains mainly b strands while a-helices form a and hydrogen bonds with residues located in the G-loop between
major part of the C-terminal region (19–21). b1 and b2 (Figure 2) (20, 22). The b- and g-phosphates in concert
with an aspartate residue and two water molecules form
Structure of CDK9 coordination bonds with a cationic Mg+2 cofactor. The aspartate
The N-terminal lobe of CDK9, which spans from amino acid residue involved in this process (Asp167 in CDK9, Asp145 in
residue 16 to 108, comprises five b structures (b1-5) and one a CDK2) belongs to a ‘DFG’ motif located in a loop between b8 and
helix (aC) (Figure 2) (22). The C-terminal lobe (residues 109- b9 (Figure 2) (18, 20, 22).
330) is composed of seven a-helices (aD-J) and four b strands The Substrate Recognition Motif is located in the cleft
(b6-9) (22). The interaction between CDK9 and cyclin T1 occurs between the N- and C- lobes in close proximity to the g-
mainly through the aC helix located on the N-terminal lobe phosphate of ATP (20). In general, CDKs have a strong
(Figures 2 and 4). This helix contains a peptide sequence highly preference for substrate motifs which have a proline residue
conserved across CDKs (PITALRE in CDK9) and serves as a site immediately flanking a phospho-Ser or phospho-Thr residue (i.e.
for interaction with cyclin during the activation of CDKs (22). Ser/Thr-Pro-X-Arg/Lys) (4). This is ascribed to the presence of a
The Adenosine Triphosphate (ATP) Binding Motif forms a hydrophobic pocket (created by the interaction between Val190
cleft between the N- and C-terminal lobes and is highly conserved and Arg195 of CDK9) that can only accommodate a proline
among CDKs (Figures 2 and 3) (22). In this site, the adenine residue at the +1 position relative to the phosphorylation site
moiety of ATP is inserted deep into the cleft and the phosphate (22). While the recognition motif is universal for the CDK
groups are positioned toward the exterior (18). The hydrophobic family, subtle differences exist between members in their
pocket harboring the adenine moiety is located between the b- substrate preference, depending on the stringent requirement
sheets of the N lobe and a hinge region loop which connects the of a specific residue at the +3 position (4). This has been
two lobes (20, 22). In this region, the ATP adenine nitrogen atoms, exemplified with the substrate recognition differences between
N6 and N1, form hydrogen bonds with the main chain oxygen and CDK2 and CDK9, where the latter displayed a strict requirement
nitrogen of Asp104 and Cys106 residues, respectively (22). In for the Ser/Thr-Pro-X-Arg/Lys consensus (23).
addition to hydrogen bonds, multiple interactions of the purine The residues of the Catalytic Loop are highly conserved
ring with aliphatic and aromatic residues of the hinge region also among protein kinases suggesting a similar catalytic
Frontiers in Oncology | www.frontiersin.org 3 May 2021 | Volume 11 | Article 678559
