Anshabo et al.

CDK9 in Cancer

Structure of Cyclins T and K

In general, cyclins are characterized by the presence of two
similar regions, each having five a-helices and a short ending
helix (N- or C- terminal helices). Each stalk of helices forms a
separate canonical cyclin box, generally composed of
approximately 100 conserved amino acid residues each. The
two regions are arranged around central helices in an antiparallel
fashion, forming a rigid structure that is liable to minor
conformational change during binding to a kinase protein (22).
Although both the N- and C- terminal regions of cyclins make
contact with the kinase protein, interactions leading to activation
of the kinase occur mainly through the N-terminal cyclin box
region (4, 19). Despite their similarity in structure and sequence,
major differences in regions outside of the cyclin box between
those cyclins involved in cell cycle control (e.g., cyclins A, B, E)
and those in transcription (e.g. cyclins T, H, C, K) are observed.
Notably, there is a clear variation in the length and orientation of
the short-ending helices (22).
The major cyclin partner of CDK9, cyclin T (‘T’ named after
the first letter of HIV TAT), has a close similarity to cyclin C and
cyclin H (15, 17). Three cyclin T members are known, namely T1
(726 residues), T2a (663 residues), and T2b (730 residues), which
have a high degree of identity (81%) in their cyclin box region
(16). HIV TAT interacts only with cyclin T1 in complex with
CDK9 to mediate HIV transcription (25, 26). Human cyclin T1
(Figure 7B) contains a recognition motif for TAT-
Transactivation Response Element (TAR) complex (TRM,
FIGURE 2 | The protein structure of monomeric CDK9 (Protein Data Bank:
residues 254-272) (26) found downstream of the N-terminal
3BLQ). The bilobal CDK9 structure is dominated by N-terminal b-sheets (1-4 are
shown) and C-terminal a-helices (D-H are shown). The C-terminal also contains cyclin box (residues 1-263), a putative coiled-coil motif (residues
b-sheets (6-9, not shown). The two lobes are connected by a hinge region (green) 379–430), histidine-rich motif (residues 506–530) (27, 28), and a
that binds the adenine moiety of adenosine triphosphate (ATP). The N-terminus C-terminal PEST (Pro-Glu-Ser-Thr) sequence (residues 709–
contains an aC helix and glycine-rich loop (G-loop, purple) which binds cyclin and 726) (29). CDK9 also forms a complex with cyclin K, which
ATP, respectively. The C-terminus comprises the catalytic loop (yellow), T-loop
(brown), and DFG motif that binds Mg+2. The threonine residue (Thr186) involved
displays CTD kinase activity, despite the fact that cyclin K only
in CDK9 activation is found in the T-loop structure. shares 29% identity with cyclin T at the amino acid level (30).
Nonetheless, the CDK9 interaction site is conserved among
cyclin T and K, explaining their similar modes of
mechanism (Figures 2 and 3) (24). The main mechanism interaction (31).
involves transformation of the hydroxyl group of the Ser or
Thr residue on the substrate into a nucleophile capable of
attacking the g-phosphate of ATP (24). A conserved aspartate
Interactions Between CDK9 and Cyclin T1
Most of the binding between CDK9 and cyclin T1 involves
(Asp149 in CDK9) facilitates this by acting as a general base that
interactions between the H3, H4, and H5 helices of the cyclin and
helps align the substrate oxygen (22, 24). Two additional
the aC helix and b4 strand of the CDK (Figures 2 and 4) (22).
residues, namely Lys151 and Thr165, have been suggested to
The H5 helix interacts with the aC helix and enforces an active
play a secondary role by orientating the substrate (22).
conformation. While this mechanism is common across CDKs,
T-Loop: When cyclin is not bound, the catalytic cleft is
in contrast to most other CDKs, the N-terminal short helix (HN)
completely blocked by a C-terminal loop named the T-loop or
in cyclin T1 makes no contact with CDK9, which gives forth to a
activation segment (Figures 2 and 3) (20). This conformation
more solvent-exposed kinase surface.
hinders critical interactions between different residues and the
non-transferable phosphates of ATP vital for locking ATP in a
catalytically favorable position. During activation, binding of cyclin
physically pulls the T-loop outward from the catalytic cleft and BIOLOGICAL FUNCTIONS OF P-TEFB
exposes a threonine residue found in the loop (Thr186 in CDK9 and
Thr160 in CDK2; Figure 3) (4, 20, 22). The phosphorylation of this Control of Transcriptional Elongation
residue stabilizes the T-loop in an open position, as phospho- and Termination
Thr186 coordinates the formation of an intramolecular hydrogen Normal cellular growth and development are dependent on
bonding network containing Arg148 and Arg172, resulting in a fully efficient and intricate regulation of gene expression. This
active kinase protein (22). regulation primarily occurs during transcription, which is the

Frontiers in Oncology | www.frontiersin.org 4 May 2021 | Volume 11 | Article 678559