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Lotz et al. Cancer Drug Resist 2020;3:149-60 I http://dx.doi.org/10.20517/cdr.2019.114 Page 151

Aberrant PTM are often found in cancer cells and are one of their distinguishing features [11,12] . The
earliest studies of PTM in Top2 proteins date back to the 1990s with the first report of a phosphorylation
[13]
of purified Top2a from mouse cells . Since then, the identification and characterization of PTM have
[14]
been accelerated by the development of proteomic techniques . Because of their relevance in cancer
therapy, Top2a PTM have been characterized mostly in cancer cell lines, while the extent of PTM in the
homeostasis of normal cells has been somewhat neglected . Recently, we identified the phosphorylation
[15]
and acetylation sites in human Top2a overproduced in the yeast S. cerevisiae and in a hamster mammalian
cell line (BHK21), thus providing a basal landscape of the modifications for Top2a isoform .
[16]

In this review, we analyze the different types of modifications affecting the human Top2a in normal and
cancer cells with a structure-function perspective. We also overview the relationships among the regulation
of Top2a by PTM, the level of PTM in cancer cells, and the resistance to anti-Top2 therapeutic compounds.

PHOSPHORYLATIONS
Phosphorylation is the most studied modification of Top2 due to its importance in the regulation of the
enzyme during the cell cycle and was shown to influence several aspects of Top2a function. The level of
[17]
phosphorylation reaches a maximum peak at G2/M phase along with the level of Top2a expression .
Several studies have reported that Top2a catalytic activity is modulated by phosphorylations [18,19] .

The majority of phosphorylations were identified in the Top2a CTD in normal and cancer cells [Figure 2].
The CTD is important for the proper subcellular localization of the enzyme as its deletion leads to Top2
[20]
mislocalization . In particular, the phosphorylation of Top2a Ser1213 is important for its relocalization
[21]
from the chromosome arms to the centromere . Phosphorylation by Casein Kinase II (CKII), Protein

Kinase C (PKC), or Extracellular signal Regulated Kinase II enhanced the decatenation and the relaxation
activities, of both human and Drosophila Top2a [17,19,22] . Inversely, incubation of Top2a with the kinase PKCζ
[23]
inhibits the decatenation activity in vivo and in vitro Phosphorylation of Ser1106 by Casein Kinase I was
.
shown to regulate decatenation since its mutation to alanine slows down the decatenation and cleavage
reactions [18,24] . However, the presence of a phosphorylated residue cannot always be directly correlated
[25]
with the modulation of the enzyme activity . Substitution of Ser1525 with an alanine does not affect
decatenation activity, despite being a major substrate for phosphorylation by CKII, Polo-like kinase I, and
p38g [26-29] .

In addition to the regulation of Top2a catalytic activity, phosphorylations appear to be important for the
recruitment of protein partners in a chromatin context. In HeLa cells, the methylated tail of Histone 3 was
[30]
shown to interact with the Top2a chromatin tether, a 30-amino-acid sequence at the end of the CTD . In
this region, three phosphorylations could be identified in cancer cells and only one thus far in normal cells,
including phosphorylation on Ser1525, which could modulate binding of Top2a to the nucleosome [Figure 2].

In the conserved catalytic domains of Top2a, three phosphorylation sites in the ATPase, topoisomerase-
primase domain (TOPRIM), and the coiled-coil domains were detected in normal cells [Figure 3]. Ten
additional phosphorylations were identified in cancer cells in the GHKL and transducer domains and in
the N-terminal arm closing the dimer [Figure 3]. Phosphorylation of Ser29 by PKC in the N-terminal arm
[17]
of the ATPase domain stimulates the DNA relaxation activity in vitro . Phosphorylations at this position
could impact the allosteric movements of the ATPase domain and the dimeric interface.

The overall level of phosphorylation was shown to modulate the ATPase activity of Top2a as well as the
cleavage/religation reaction in vitro [22,31,32] . More phosphorylations can be found in cancer cells in the
TOPRIM and winged helix domain (WHD) domains, and lining the coiled-coil region [Figures 2 and 3B].
Although less abundant, six phosphotyrosines in the Top2a protein were identified in cancer cells [Figure 3B].