CKS Proteins Promote Checkpoint
Recovery by Stimulating Phosphorylation
of Treslin
Ruiling Mu,
a
John Tat,
a
Robert Zamudio,
a
Yaoyang Zhang,
a
*
John R. Yates III,
a
Akiko Kumagai,
b
William G. Dunphy,
b
Steven I. Reed
a
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA
a
; Division of Biology
and Biological Engineering, California Institute of Technology, Pasadena, California, USA
b
ABSTRACT
CKS proteins are small (9-kDa) polypeptides that bind to a subset of the
cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2,
share an overlapping function that is essential for early development. However, both
proteins are frequently overexpressed in human malignancy. It has been shown that
CKS protein overexpression overrides the replication stress checkpoint, promoting
continued origin firing. This finding has led to the proposal that CKS protein-dependent
checkpoint override allows premalignant cells to evade oncogene stress barriers,
providing a causal link to oncogenesis. Here, we provide mechanistic insight into how
overexpression of CKS proteins promotes override of the replication stress check-
point. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphory-
late the key replication initiation protein treslin
in vitro
. Furthermore, stimulation of
treslin phosphorylation does not occur by the canonical adapter mechanism demon-
strated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mu-
tants are capable of stimulating treslin phosphorylation. This effect is recapitulated
in vivo
, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and
overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-
dependent dephosphorylation of treslin. Finally, we provide evidence that the role
of CKS protein-dependent checkpoint override involves recovery from checkpoint-
mediated arrest of DNA replication.
KEYWORDS
CKS protein, treslin, treslin phosphorylation, replication stress
checkpoint, checkpoint recovery
C
KS proteins were discovered in budding yeast and fission yeast based on genetic
interactions with
CDK1
mutants (
cdc28
in budding yeast and
cdc2
in fission yeast)
(
1
,
2
). Based on homology, similar proteins were identified in a variety of eukaryotes,
including
two paralogs in vertebrates, Cks1 and Cks2 (
3
). Although these small con-
served
proteins were shown, shortly after their discovery, to bind to a subset of
cyclin-dependent kinases (CDKs) (
4
), their function(s) remained obscure until recently.
The
crystallographically determined geometry of Cks1 bound to Cdk2 suggested an
adaptor function, promoting targeting of the kinase to substrates (
5
). The fact that CKS
proteins
possess a phosphate binding site has led to the hypothesis that CKS proteins
can tether CDKs to substrates already primed by phosphorylation for efficient subse-
quent multiphosphorylation. Indeed, this mechanism has been borne out for some
substrates, such as the yeast CDK inhibitors Sic1 and Far1 (
6
,
7
), and is probably the case
for
a variety of other CDK substrates in vertebrates, such as Cdc27, Cdc25, Wee1, and
Myt1 (
8
,
9
). However, a number of other well-characterized adapter functions have
been
attributed to CKS proteins, all presumably stemming from their ability to form
relatively high-affinity complexes with CDKs. CKS proteins are critical for efficient
docking of Cdk2-cyclin A complexes to the ubiquitin ligase anaphase-promoting
Received
21 June 2017
Returned for
modification
10 July 2017
Accepted
11 July
2017
Accepted manuscript posted online
24 July
2017
Citation
Mu R, Tat J, Zamudio R, Zhang Y,
Yates JR, III, Kumagai A, Dunphy WG, Reed SI.
2017. CKS proteins promote checkpoint
recovery by stimulating phosphorylation of
treslin. Mol Cell Biol 37:e00344-17.
https://doi
.org/10.1128/MCB.00344-17
.
Copyright
©
2017 American Society for
Microbiology.
All Rights Reserved
.
Address
correspondence to Steven I. Reed,
sreed@scripps.edu.
*
Present address: Yaoyang Zhang,
Interdisciplinary Center for Biology and
Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences,
Shanghai, China.
RESEARCH ARTICLE
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complex/cyclosome (APC/C) for the metaphase ubiquitin-dependent proteolysis of
cyclin A (
10
). The paralog Cks1 is essential for the binding of the SCF ubiquitin ligase
specificity
factor Skp2 to the CDK-bound inhibitor p27
Kip1
for ubiquitylation and
degradation (
11
,
12
). Perhaps the most unexpected roles for CKS proteins are in the
realm
of transcriptional regulation. In budding yeast, it was found that the Cks1-Cdk1
complex has a kinase-independent role in nucleosome eviction during rapid transcrip-
tional induction (
13
,
14
). This function requires the Paf1 elongation complex and the
19S
proteasome particle (
15
). In mammalian cells, the essential redundant function of
Cks1
and Cks2 is the expression of mRNAs encoding Cdk1, cyclin B1, and cyclin A2 (
16
).
Although
the molecular mechanism remains to be elucidated, it may involve a chro-
matin remodeling function analogous to what has been observed in yeast.
In addition to their normal cellular roles, CKS proteins are likely to have roles in
oncogenesis. Cks1 and/or Cks2 are frequently overexpressed in a broad spectrum of
malignancies (
17–30
). It has been hypothesized that Cks1 overexpression is linked to its
role
in degradation of the CDK inhibitor p27
Kip1
, as low p27 levels have been associated
with aggressive malignancy in various types of cancer. However, attempts to correlate
high Cks1 levels with low p27 levels in tumors have given mixed results (
31–33
).
Another
potential insight into the role of Cks proteins in oncogenesis comes from
the observation that Cks1 or Cks2, when overexpressed, overrides the replication stress
checkpoint (
34
). Whereas triggering of the replication stress checkpoint normally
prevents
subsequent replication origin firing, cells overexpressing either Cks1 or Cks2
continue to fire origins, even though checkpoint signaling is intact. This observation led
us to speculate that CKS protein overexpression allows premalignant cells to evade
DNA damage checkpoint barriers triggered as the first line of defense in response to
activated or overexpressed oncoproteins. Indeed, we observed that there was a strong
correlation in breast tumors between overexpression of cyclin E (
34
), an oncoprotein
that
causes replication stress, and either Cks1 or Cks2. The replication stress checkpoint
detects single-stranded DNA resulting from stalled or collapsed replication forks and
signals to Cdk2 by promoting the degradation of the CDK phosphatase CDC25A
(
35–37
). As a result, Cdk2 accumulates in its tyrosine 15-phosphorylated inactive form
(
38
). The link between Cdk2 activity and origin firing is likely to be the protein treslin
(
39
,
40
). The assembly of an active replicative helicase requires the CDK-dependent
phosphorylation
of treslin on serine 1000 (S1000). Phosphorylated treslin then binds to
BRCT domains I and II of the protein TopBP1, leading to the recruitment of the initiator
protein Cdc45. This is a highly conserved pathway, where in yeast Sld3 the treslin
ortholog binds to Dbp11, the TopBP1 ortholog, in a CDK-dependent manner to initiate
replication (
41
,
42
). Checkpoint-mediated inhibition of Cdk2 presumably leads to
reversal
of these events, precluding subsequent origin firing.
Since CKS protein overexpression permits origin firing in the context of an active
replication stress checkpoint, we sought to determine if there is a link between CKS
protein function and treslin phosphorylation. We found that both Cks1 and Cks2 greatly
enhance the efficiency of treslin phosphorylation by Cdk2
in vitro
in a reconstituted
reaction using purified proteins as well as in cultured cells. Interestingly, the stimulation
of treslin phosphorylation does not occur by a mechanism whereby the CKS protein
serves as a canonical CDK substrate adapter, as previously reported, but rather by
improving the efficiency of CDK-treslin interaction either by remodeling treslin or by
serving as a noncanonical Cdk2 adapter. Furthermore, we provide evidence that the
function of CKS-enhanced phosphorylation of treslin entails recovery from the replica-
tion stress checkpoint.
RESULTS
Both Cks1 and Cks2 stimulate phosphorylation of treslin by Cdk2.
In
order to
determine whether Cks1 and/or Cks2 has a role in treslin phosphorylation on serine
1000 (S1000), we developed an
in vitro
system using purified proteins. As a surrogate
substrate for treslin, we used a glutathione
S
-transferase (GST) fusion to a 23-amino-
acid (aa) treslin peptide centered around S1000 [aa 989 to 1011; termed treslin
Mu et al.
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(989-1011)] (
40
)(
Fig. 1A
). We purified active cyclin E-Cdk2 from an
Escherichia
coli
strain
that also expresses the yeast Cak1 kinase, which phosphorylates the CDK T-loop-
producing active enzyme (
43
). Cks1 and Cks2 were also purified from
E.
coli
. Phosphor-
ylation of the treslin substrate after incubation with Cdk2 was detected using a
phosphoepitope-specific antibody (
40
). GST-treslin and Cks1/2 were detected by amido
black
staining of blots. Cyclin E-Cdk2 was capable of phosphorylation of the treslin
peptide without any additional proteins. However, addition of Cks1 or Cks2 stimulated
treslin phosphorylation by 10- to 20-fold (
Fig. 1B
and
C
). Cks1 also stimulated phos-
phorylation
of a larger treslin polypeptide fused to GST (aa 893 to 1257) (
Fig. 1D
)(
40
).
In
order to rule out that the stimulation of treslin peptide phosphorylation was due to
an interaction between CKS protein and the GST tag, we carried out a parallel
experiment using a pure synthetic peptide [treslin (997-1008)] and determined the
degree of phosphorylation by mass spectroscopy (MS). Cks1 increased phosphorylation
of this peptide by Cdk2 by 10-fold (
Fig. 2
), similar to what was observed using GST
fusion
and Western blotting. Therefore, CKS proteins stimulate treslin peptide phos-
phorylation by interacting directly with treslin sequences.
Dose-dependent phosphorylation of treslin peptide.
A kinase assay, as described
above, was performed with Cks1 concentrations ranging from 1.85 nM to 3.7
M(
Fig.
3A
). The reaction mixture concentrations of Cdk2 and GST-treslin peptide were 17 nM
and
1.1
M, respectively. As can be seen in
Fig. 3A
, the phosphorylation of treslin
peptide
saturates at stoichiometry of Cks1 approximately equivalent to that of GST-
treslin peptide and well in excess of that of Cdk2. On the other hand, the 50% effective
concentration (EC
50
) of Cks1-mediated stimulation of treslin phosphorylation is 90 nM
(
Fig. 3B
), similar to the previously determined dissociation constant (
K
d
) of Cks1 binding
to Cdk2 (77 nM) (
5
). Therefore, these data are consistent with Cks1 functioning through
binding
to Cdk2 or as a stoichiometric substrate adapter. In order to investigate this
finding further, we determined the smallest treslin peptide capable of CKS protein-
stimulated phosphorylation. An 8-amino-acid peptide (aa 997 to 1004) fused to GST
exhibited CKS-enhanced phosphorylation similar to that of treslin (989-1011) (
Fig. 3C
).
Considering
that Cks1 has been shown to bind to the C-lobe of Cdk2 distal from the
A
Treslin
(989-1011)
:
DPGPDIGVVEESPEKGDEIGLRR
B
-EK2 +EK2 +EK2
-Cks1 -Cks1+Cks1 (1.85
μ
M
)
pS1000
GST-Treslin
(989-1011)
Cks1
C
- Cks1 Cks2
pS1000
GST-Treslin
(989-1011)
Cks1/2 (3.7
μ
M
)
D
pS1000
GST-Treslin
(893-1257)
FIG 1
Cks1 and Cks2 stimulate treslin phosphorylation
in vitro
. (A) GST-treslin substrate. A 23-amino-acid
peptide centered around treslin S1000 fused to GST was used as a Cdk2 substrate for most
in vitro
phosphorylation experiments described. (B) Stimulation of GST-treslin phosphorylation by Cks1.
In vitro
kinase reactions were carried out using a 23-amino-acid peptide flanking the S1000 phosphorylation site
of treslin fused to GST. Each 30-
l reaction mixture contained 17 nM recombinant cyclin E/Cdk2, 1.1
M
GST-treslin, and the indicated concentration of Cks1. Phosphorylation of the treslin substrate was
detected after SDS-PAGE and Western blotting using an antibody that recognizes phosphorylated S1000
of treslin. Cks1 and GST-treslin were detected by amido black staining. (C) Cks1 and Cks2 both stimulate
phosphorylation of GST-treslin. Reaction mixtures like those described for panel B were set up with 3.7
M Cks1 or Cks2. (D) Cks1 stimulates Cdk2-mediated phosphorylation of a larger fragment of treslin
fused to GFP (aa 893 to 1257). Reaction mixtures were like those described for panel B but with 0.185
M Cks1.
CKS Proteins Stimulate Treslin Phosphorylation
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active site (
5
), it would be impossible for an 8-amino-acid peptide with an internal
phosphorylation
site to bind simultaneously to the active site and to CDK-bound Cks1.
We then carried out an experiment to determine whether Cks1 or Cks2 could form a
complex directly with treslin in the absence of Cdk2. Thermal shift analysis was carried
out on CKS protein-treslin peptide mixtures. Incubation with synthetic treslin peptide
FIG 2
Cks1 enhances phosphorylation of a small synthetic treslin peptide. Kinase reactions were as described in the legend to
Fig. 1
. A synthetic peptide
corresponding
to treslin amino acids 997 to 1008 was incubated with buffer only, with cyclin E-Cdk2 alone, or with cyclin E-Cdk2 and Cks1 (1.1
M). The three
mixtures were infused into an Orbitrap mass spectrometer using the ESI method, and both MS1 and MS2 were recorded. MS peaks representing unmodified
peptide and phosphopeptide are indicated.
A
1.85 nM
3.7
μ
M
Cks1
GST-Treslin
(989-1011)
Cks1
pS1000
B
989-1011
997-1008
997-1006
997-1004
989-1011
997-1008
997-1006
997-1004
989-1011
Cks1
Cks2
GST-Treslin
Cks1/2
pS1000
C
FIG 3
CKS proteins stimulate treslin phosphorylation by a noncanonical mechanism. (A) Dose-dependent Cks1
stimulation of GST-treslin (989-1011) phosphorylation. The kinase assay was performed as described in the legend
to
Fig. 1
. Cks1 concentrations ranged from 1.85 nM (left) to 3.7
M (second from the right). The area on the far right
has no Cks1. (B) Quantification of data shown in panel A using Image J and GraphPad Prism software. arb, arbitrary;
conc, concentration. (C) CKS proteins stimulate phosphorylation of 8-amino-acid peptides flanking the treslin S1000
phosphorylation site. GST fusions corresponding to 23-, 12-, 10-, and 8-amino-acid treslin peptides were incubated
with Cks1 or Cks2, as indicated, and cyclin E/Cdk2 as described in the legend to
Fig. 1
. The lane on the far right
corresponds
to a reaction without CKS protein.
Mu et al.
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