Content uploaded by Kavita Shah
Author content
All content in this area was uploaded by Kavita Shah on Mar 28, 2019
Content may be subject to copyright.
Mammalian cell overexpression and siRNA depletion
Yip3 was cloned into the mammalian vector pcDNA3.1. HEK293 cells were transfected
with pcDNA3.1 human Yip3 or pcDNA3.1, collected 42 h later,lysed by osmotic shock and
shearing, and membranes were purified by ultracentrifugation. Membrane recruitment
was as published
6,7
in buffer plus 100 mM (NH
4
)
2
SO
4
, 100
m
M GTP
g
S, 0.1 mg ml
21
BSA,
protease inhibitor cocktail (0.34 units ml
21
aprotinin, 0.01 mg ml
21
leupeptin, 1
m
M
pepstatin A), 0.2 mM DTT and ATP regeneration system
6,7
. Rab9–GDI complex (300 ng,
4.1 pmol) and membranes were mixed in 250
m
l for 40 min at 37 8C. Ice-cold buffer
(64 mM HEPES-KOH, pH 8.0, 100 mM NaCl, 8 mM MgCl
2
, 2 mM EDTA; 0.5 ml) was
added and each reaction was then layered over 0.4M sucrose þice-cold buffer and spun
for 10 min at 342,000g,28C in a TLA-100.2 rotor (Beckman). Pellets were analysed by
12.5% SDS–PAGE and anti-Rab9 immunoblot.
HeLa S-3 cells were transfected with 0.13
m
M siRNA duplex (sense 5 0-(GCUUGUG
CUCUUUGGCCGA)d(TT)-3 0; Dharmacon) using Oligofectamine (Invitrogen). After
77 h, cells were lysed in 25mM HEPES pH 7.3 with protease inhibitors by 10 passages
through a 25G needle. Extracts were spun 30s at 1,000 g; supernatants were centrifuged
15 min, 321,000 gin a TLA 120.1 rotor (Beckman). The membrane pellet was washed in
PBS.
Received 16 July; accepted 8 September 2003; doi:10.1038/nature02057.
1. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117
(2001).
2. Segev, N. Ypt and Rab GTPases: Insight into functions through novel interactions. Curr. Opin. Cell
Biol. 13, 500–511 (2001).
3. Pfeffer, S. R. Rab GTPases: Specifying and deciphering organelle identity and function. Trends Cell
Biol. 11, 487–491 (2001).
4. Sasaki, T. et al. Purification and characterization from bovine brain cytosol of a protein that inhibits
the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like
GTP-binding protein. J. Biol. Chem. 265, 2333–2337 (1990).
5. Pfeffer,S. R., Dirac-Svejstrup, A. B. & Soldati, T.Rab GDP dissociation inhibitor: Putting Rab GTPases
in the right place. J. Biol. Chem. 270, 17057–17059 (1995).
6. Soldati, T., Shapiro, A. D., Svejstrup, A. B. & Pfeffer, S. R. Membrane targeting of the small GTPase
Rab9 is accompanied by nucleotide exchange. Nature 369, 76–78 (1994).
7. Soldati, T.,Rancano, C., Geissler, H. & Pfeffer, S. R. Rab7 and Rab9 are recruited onto late endosomes
by biochemically distinguishable processes. J. Biol. Chem. 270, 25541–25548 (1995).
8. Ullrich, O., Horiuchi, H., Bucci, C. & Zerial, M. Membrane association of Rab5 mediated by GDP-
dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 368, 157–160 (1994).
9. Shapiro, A. D. & Pfeffer, S. R. Quantitative analysis of the interactions between prenyl Rab9, GDP
dissociation inhibitor-alpha, and guanine nucleotides. J. Biol. Chem. 270, 11085–11090 (1995).
10. Dirac-Svejstrup, A. B., Sumizawa, T. & Pfeffer, S. R. Identification of a GDI displacement factor that
releases endosomal Rab GTPases from Rab-GDI. EMBO J. 16, 465–472 (1997).
11. Yang, X., Matern, H. T. & Gallwitz, D. Specific binding to a novel and essential Golgi membrane
protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p. EMBO J. 17, 4954–4963
(1998).
12. Calero, M. & Collins, R. N. S. cerevisiae Pra1p/Yip3 interacts with Yip1p and Rab proteins. Biochem.
Biophys. Res. Commun. 290, 676–681 (2002).
13. Calero, M., Winand, N. J. & Collins, R. N. Identification of the novel proteins Yip4pand Yip5p as Rab
GTPase interacting factors. FEBS Lett. 515, 89–98 (2002).
14. Matern, H. et al. A novel Golgi membrane protein is part of a GTPase-binding protein complex
involved in vesicle targeting. EMBO J. 19, 4485–4492 (2000).
15. Martincic, I., Peralta, M. E. & Ngsee, J. K. Isolation and characterization of a dual prenylated Rab and
VAMP2 receptor. J. Biol. Chem. 272, 26991–26998 (1997).
16. Bucci, C., Chiariello, M., Lattero, D., Maiorano, M. & Bruni, C. B. Interaction cloning and
characterization of the cDNA encoding the human prenylated Rab acceptor (PRA1). Biochem.
Biophys. Res. Commun. 258, 657–662 (1999).
17. Hutt, D. M., Da-Silva, L. F., Chang, L. H., Prosser,D. C. & Ngsee, J. K. PRA1 inhibits the extraction of
membrane-bound Rab GTPase by GDI1. J. Biol. Chem. 275, 18511–18519 (2000).
18. Abdul-Ghani, M., Gougeon, P. Y., Prosser, D. C., Da-Silva, L. F. & Ngsee, J. K. PRA isoforms are
targeted to distinct membrane compartments. J. Biol. Chem. 276, 6225–6233 (2001).
19. Lin, J., Liang, Z., Zhang, Z. & Li, G. Membrane topography and topogenesis of prenylated Rab
acceptor (PRA1). J. Biol. Chem. 276, 41733–41741 (2001).
20. Figueroa, C., Taylor, J. & Vojtek, A. B. Prenylated Rab acceptor protein is a receptor for prenylated
small GTPases. J. Biol. Chem. 276, 28219–28225 (2001).
21. Otte, S. et al. Erv41p and Erv46p: New components of COPII vesicles involved in transport between
the ER and Golgi complex. J. Cell Biol. 152, 503–518 (2001).
22. Barrowman, J., Wang, W., Zhang, Y. & Ferro-Novick, S. TheYip1p-Yif1p complex is required for the
fusion competence of endoplasmic reticulum-derived vesicles. J. Biol. Chem. 278, 19878–19884
(2003).
23. Tang, B. L. et al. A membrane protein enriched inER exit sitesinteracts with COPII. J. Biol. Chem. 276,
40008–40017 (2001).
24. Soldati, T., Shapiro, A. D. & Pfeffer, S. R. Reconstitution of the endosomal targeting of rab9 protein
using purified, prenylated rab9 protein as a complex with GDI. Methods Enzymol. 257, 253–259
(1995).
25. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. A. An improved assay for nanomole amounts
of inorganic phosphate. Anal. Biochem. 100, 95–97 (1979).
26. Fairbanks, G., Steck, T.L. & Wallach, D.F. H. Electrophoretic analysis of the major polypeptides of the
human erythrocyte membrane. Biochemistry 22, 2606–2617 (1971).
Supplementary Information accompanies the paper on www.nature.com/nature.
Acknowledgements We thank F. Barr and T. Meyer for providing Rab2 and Rab1A plasmids,
respectively, and B. Stafford and C. Melton for technical assistance. This work was supported by
grants from the NIH and the American Heart Association. U.S. and D.A. were postdoctoral
fellows of the Swedish Foundation for International Cooperation in Research and Higher
Education and the Leukemia and Lymphoma Society, respectively.
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to S.R.P. (pfeffer@stanford.edu).
..............................................................
Targets of the cyclin-dependent
kinase Cdk1
Jeffrey A. Ubersax
1
, Erika L. Woodbury
1
, Phuong N. Quang
1
,
Maria Paraz
1
, Justin D. Blethrow
1,2
, Kavita Shah
3
, Kevan M. Shokat
2
& David O. Morgan
1
1
Departments of Physiology and Biochemistry &Biophysics, and
2
Department of
Cellular and Molecular Pharmacology, University of California, San Francisco,
California 94143, USA
3
Genomics Institute of the Novartis Foundation, San Diego, California 92121,
USA
.............................................................................................................................................................................
The events of cell reproduction are governed by oscillations in the
activities of cyclin-dependent kinases (Cdks)
1
. Cdks control the
cell cycle by catalysing the transfer of phosphate from ATP to
specific protein substrates. Despite their importance in cell-cycle
control, few Cdk substrates have been identified
2
. Here, we
screened a budding yeast proteomic library for proteins that
are directly phosphorylated by Cdk1 in whole-cell extracts. We
identified about 200 Cdk1 substrates, several of which are
phosphorylated in vivo in a Cdk1-dependent manner. The iden-
tities of these substrates reveal that Cdk1 employs a global
regulatory strategy involving phosphorylation of other regulat-
ory molecules as well as phosphorylation of the molecular
machines that drive cell-cycle events. Detailed analysis of these
substrates is likely to yield important insights into cell-cycle
regulation.
We developed methods for the systematic identification of Cdk
substrates in the budding yeast Saccharomyces cerevisiae, a simple
eukaryote in which the cell cycle is controlled by a single Cdk, Cdk1
(or Cdc28). We used a recently developed approach that allows the
specific labelling of the substrates of a single kinase in a cell
extract
3,4
. This method involves the mutation of a conserved
bulky residue in the ATP-binding pocket to a glycine or alanine.
The resulting analogue-sensitive (as) kinase is able to bind bulky
ATP analogues that fit into the mutant-binding pocket but cannot
bind to wild-type kinases. Importantly, the mutation is deep in the
ATP-binding pocket, far from the protein substrate binding site, and
is not expected to alter the protein substrate specificity of the
protein kinase
5
. The addition of radiolabelled ATP analogue to a
Table 1 Nucleotide specificity of Cdk1-as1
Kinase Nucleotide K
m
(
m
M)
k
cat
(min
21
)
k
cat
/K
m
(
m
M
21
min
21
)
.............................................................................................................................................................................
Cdk1zClb2 ATP 35 132 3.73
Cdk1-as1zClb2 ATP 322 21.3 0.07
Cdk1zClb2 N
6
-(benzyl) ATP .1,000 NM NM
Cdk1-as1zClb2 N
6
-(benzyl) ATP 1.5 13.6 9.07
.............................................................................................................................................................................
Active cyclin-Cdk complexes were formed by the addition of excess MBP-Clb2 to purified Cdk1-
His
6
or Cdk1-as1-His
6
, and their ability to phosphorylate histone H1 at different N
6
-(benzyl) ATP or
ATP concentrations was measured to generate K
m
and k
cat
values. NM, V
max
not measurable at
1 mM nucleotide.
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature 859
© 2003 Nature Publishing Group
cell extract containing a single as-kinase therefore leads to the
specific labelling of the direct substrates of that kinase.
In previous work, we constructed an analogue-sensitive version
of yeast Cdk1, Cdk1-as1, by replacing phenylalanine 88 with
glycine
6
. We showed that Cdk1-as1 is functional in vivo and is
uniquely sensitive to a bulky chemical inhibitor that interacts with
the enlarged ATP-binding site. In the present work, we measured
Cdk1-as1 activity with the bulky ATP analogue N
6
-(benzyl) ATP.
As predicted, Cdk1-as1 (in a complex with the mitotic cyclin
Clb2) exhibited a high affinity for the ATP analogue, whereas
wild-type Cdk1 had no measurable activity with this substrate
(Table 1). Cdk1-as1 was approximately 130-fold more
active towards N
6
-(benzyl) ATP than ATP, that is,
ðkcat=KmÞN62ðbenzylÞATP =ðkcat=KmÞATP ¼130 (Table 1). Thus, the
substitution of a glycine for a phenylalanine in the active site of
Cdk1 results in a mutant that displays high affinity and selectivity
for N
6
-(benzyl) ATP.
Concentrated cell extracts, prepared by gentle detergent lysis of
yeast spheroplasts, were incubated with radiolabelled N
6
-(benzyl)
ATP. Few proteins were labelled, indicating that wild-type kinases
in the extract were not using the ATP analogue at a significant rate
(Fig. 1, lane 1). We then added purified Cdk1-as1zClb2 complexes at
a concentration (7 nM) that resulted in levels of activity similar to
those in mitotic cell extracts. Many radiolabelled proteins were
generated (Fig. 1, lane 2). Incubation of purified Cdk1-as1zClb2
with ATP analogue in the absence of cell extract resulted in
autophosphorylation of Clb2 and other contaminants (Fig. 1, lane
3), and revealed that most of the proteins labelled in lane 2 represent
direct Cdk1 substrates in the crude cell extract.
We next developed a simple, rapid and highly sensitive approach
to scan the yeast proteome for proteins phosphorylated by Cdk1-
as1zClb2. We used a library of 6,144 yeast strains, each expressing a
unique open reading frame (ORF) with an amino-terminal fusion
to glutathione-S-transferase (GST)
7
. We reasoned that we could
perform kinase reactions with Cdk1-as1 in lysates of strains from
this library, and then purify the GST-tagged protein to determine if
it was phosphorylated.
Pilot studies revealed that tagged substrate phosphorylation was
not readily detected if reactions were performed with extracts made
from a pool of multiple GST-ORF yeast strains. It was therefore
necessary to perform separate reactions for each GST-tagged pro-
tein. Rather than performing over six thousand reactions, we
focused on several hundred candidate ORFs that seemed likely to
encode Cdk targets. As most known Cdk substrates contain mul-
tiple Cdk consensus phosphorylation sites (S/T*-P-x-K/R, where
x¼any amino acid), we first tested the 385 ORFs encoding proteins
with two or more of these sites. In addition, because cell-cycle
regulators are often controlled by transcriptional mechanisms, we
screened the 137 ORFs encoding proteins with a single Cdk
consensus site and whose transcripts are cell-cycle regulated
8
.
Finally, in an attempt to estimate the number of Cdk targets in
the entire proteome, we tested 198 randomly chosen ORFs. These
candidates encompass a total of 695 unique ORFs (due to 25
overlaps between the random 198 and the 522 candidates), which
represent about 11% of the yeast proteome.
Results from twelve representative reactions are shown in Fig. 2a.
To rank the substrates, we measured the phosphorylation of each
tagged protein (and fragments) and divided by the amount of
tagged protein present in the reaction. The amounts of phosphate
incorporated per nanogram of protein ranged over seven orders of
magnitude. The logarithms of these values were calculated and
defined as the ‘P-score’ for each tagged protein.
Of the 695 tested proteins, 360 were detectably phosphorylated
(Fig. 2b). Of these 360 proteins, 181 had P-scores of 2.0 or higher
and represent the most interesting group of substrates because they
were less-abundant proteins with high levels of phosphorylation. As
shown in Fig. 2c, 179 of the 181 top substrates were present in our
reactions at very low concentrations (,50 pM to 50 nM; see Fig. 2c
legend), suggesting that phosphorylation of these proteins was not
an artefact of high substrate concentrations. Forty of the top 181
substrates are listed in Table 2 (a complete list of results with all
tested proteins is available as Supplementary Information).
Approximately 12 proteins containing full Cdk consensus sites
have been identified previously as likely Cdk targets in budding
yeast (Table 2, marked in bold: Swi5, Sic1, Cln2, Cdh1, Far1, Gin4,
Swe1, Cdc6, Orc2, Orc6, Sld2 and Pds1)
9–20
. Ten of these proteins
had P-scores greater than 2 in our screen. Phosphorylation of the
remaining two, Cdc6 and Cln2, was apparently undetectable as a
result of the limitations of the tagged protein library (see Methods).
Thus, essentially all known Cdk targets with full consensus sites are
among the top 181 substrates we identified, which strongly supports
the validity of our approach.
Our results provide insights into mechanisms that govern sub-
strate recognition by Cdks. A small number of Cdk targets in
yeast and other species do not contain the full Cdk consensus site
(S/T*-P-x-K/R) but are instead phosphorylated at a minimal con-
sensus site containing a serine or threonine residue followed by
proline (S/T*-P)
21
. Of the 198 random proteins we tested, 16 were
phosphorylated with P-scores of greater than two. Thirteen of these
proteins contain the full consensus sequence, and the remaining
three contain multiple copies of the minimal consensus site. We
therefore suspect that the great majority of Cdk targets contain the
full consensus site. However, the presence of a full consensus site is
not sufficient for Cdk phosphorylation: we found 123 proteins with
this site that were not phosphorylated, despite being expressed at
detectable levels.
Substrate recognition by Cdks is influenced by the associated
cyclin subunit
22,23
. Although our screen was carried out with the
mitotic Cdk1-as1zClb2 complex, we found that several substrates
involved in DNA replication (including Orc6, Mcm3 and Sld2) are
Figure 1 Addition of Cdk1-as1 and radiolabelled N
6
-(benzyl) ATP to a cell extract results
in the appearance of many specific phosphoproteins. 5
m
Ci
g
-
32
P-N
6
-(benzyl) ATP
was added to 30
m
g whole yeast extract (lanes 1, 2) or buffer (lane 3) in the presence
(lanes 2, 3) or absence (lane 1) of 7 nM purified Cdk1-as1zClb2. After incubation for
30 min at room temperature (23 8C), reactions were analysed by SDS–PAGE on a 5–15%
gradient gel, and autoradiographed. Asterisk indicates autophosphorylated Clb2 bands.
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature860 © 2003 Nature Publishing Group
Cdk1
-as1
•Clb2
+ +
Cell
extract + +
(kDa)
220 -
160 -
120 -
100 -
90
-
80
-
70
-
60
-*
50
-
40
-
30
-
25
-
20
-
15
-
2 3
phosphorylated tenfold more rapidly by the S-phase Cdk1zClb5
complex (M. Loog and D.O.M., unpublished results). We therefore
believe that our screen has led to the identification of a broad range
of Cdk targets involved in multiple cell-cycle stages.
To confirm that we have identified proteins that are substrates of
Cdk1 in the cell, it is important to show that phosphorylation of
these proteins in vivo is dependent on Cdk1 activity. Traditionally,
temperature-sensitive cdk1 mutants have been used to demonstrate
that phosphorylation is Cdk1-dependent in vivo. However, inacti-
vation of these mutants takes 2 to 3 h at the restrictive temperature
and results in a cell-cycle block (typically in G1), making it difficult
to rule out indirect effects caused by the shift in cell-cycle position.
These problems can be circumvented with the cdk1-as1 strain,
in which it is possible to inhibit much of the cell’s Cdk1 activity
within 5–10 min by addition of high concentrations (5–25
m
M) of
1-NM-PP1, a small molecule that inhibits the analogue-sensitive
mutant Cdk1 without affecting other kinases in the cell
6
. The speed
of inhibition reduces the likelihood of indirect effects.
To rapidly assess the phosphorylation state of a subset of
substrates in vivo, we identified candidates that migrate on poly-
acrylamide gels as multiple bands (such mobility shifts can be a
useful indication of phosphorylation). We prepared or obtained
strains expressing epitope-tagged versions of 171 of the 181 candi-
date substrates expressed at their endogenous loci. A hundred
proteins were detectable on western blots, and approximately 35
of these proteins displayed reproducible mobility shifts. We initially
focused on Slk19, a non-essential regulator of spindle assembly and
stability. Slk19 is a substrate of separase (Esp1)
24
and migrates on
gels as a pair of band clusters, in which the lower cluster is the
carboxy-terminal separase-cleavage product. As reported pre-
viously
24
, the reduced mobility of the upper forms in each cluster
is caused by phosphorylation and is maximal in cells arrested in
S phase or mitosis (Fig. 3a), consistent with phosphorylation by
Cdk1. Treatment of cdk1-as1 cells with 1-NM-PP1 caused dephos-
phorylation of the upper Slk19 band within five minutes, arguing
that phosphorylation in vivo is Cdk1-dependent (Fig. 3b). After
30 min of treatment, the drop in mitotic cyclin levels presumably
reflects activation of the Cdh1-dependent APC, which is normally
inhibited by Cdk1
12,13
. Phosphorylation of the separase-cleavage
product was not reversed by Cdk1 inhibition, perhaps indicating
Figure 2 181 proteins are efficiently phosphorylated by Cdk1-as1zClb2.
a, Autoradiograph (top) and silver-stained gel (bottom) of reactions with twelve GST-fusion
proteins. 7 nM Cdk1-as1zClb2 and 5
m
Ci
g
-
32
P-N
6
-(benzyl) ATP were added to whole
yeast extracts expressing a single GST-tagged protein and incubated for 30 min at
room temperature, after which the GST-tagged protein was purified and analysed by
SDS–PAGE. b, Histogram of the distribution of P-scores for the 695 GST-tagged proteins
reveals 181 efficiently phosphorylated Cdk1 substrates (P-score .2, dark blue). c, Most
of the best Cdk1 substrates are present at low concentrations in our reactions.
GST-tagged proteins were grouped by their estimated protein amounts (nanograms in a
200
m
l reaction volume). If we assume a relative molecular mass of 100,000, then
1,000 ng tagged protein represents a concentration of 50 nM. The number of proteins
with P-scores above 2, between 0 and 2, or equal to zero, are shown for each group.
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature 861
© 2003 Nature Publishing Group
a
(kDa)
220-
160-
120-
100-
90-
80-
70-
60-
50-
40-
30-
25-
160
120
100
90
80
70
60
50
40
30
25
3:
0
'SI"
co
"'
:i
(/)
CJ
a.
::iE
a:
0 a.
N
CD
>-
::iE
()
()
3:
()
,.._
co
;:::
;;;
,.._
'SI"
1i:
"'
w
:3
1i:
0
"'
C\J
a: a:
>-
I
0 0
(/)
<t
a. 0 0
>-
>-
::iE
CJ
>- >-
<t
u..
b 400
(kDa)
350 335
-220
-160
U)
C 300
-120
"
iii
250
-
-100
e
a. 200
-90
0
-80
t 150
-70
.Q
100
E
:::,
-60
z 50
0
-50
0
-40
-30
-25
C 800
700
220
160
U)
600
C
"
iii
e 500
a.
120 0 400
t
100
90
.Q
E 300
:::,
80
70
z 200
60 100
0
50 ng
of
protein
40 I• P-score > 2
I
El
0 > P-score > 2
lo P-score = 0
30
25
94
0-1
1-2
2-3
3-4
4-5
5-6
6-7
P-score
1=
,-
-,-
-,_
• I
- ~
-,-
• n
11-
101-
<1
1-10
100 1,000
>1
,000 Total
61
34 55 29 2
181
0 7 47 89 36 179
156 20
90
59 10 335
that this fragment is less susceptible to phosphatases or is phos-
phorylated by other kinases.
Slk19 contains three full Cdk consensus sites (S/T*-P-x-K/R) and
three minimal Cdk consensus sites (S/T*-P). Changing all six of
these residues to alanine abolished phosphorylation of the protein
in vivo (data not shown), further arguing that Slk19 is phosphory-
lated by Cdk1 in vivo. Replacement of the endogenous SLK19 gene
with a mutant gene encoding the non-phosphorylatable mutant
resulted in a strain that appeared wild-type in all respects (cell-cycle
timing, spindle structure and sporulation), indicating that Slk19
phosphorylation at these sites is not essential for cell-cycle pro-
gression under routine laboratory conditions.
We next analysed the effects of Cdk1 inhibition on other
candidates with detectable gel mobility shifts. A fifteen-minute
treatment of asynchronous cdk1-as1 cells with 1-NM-PP1 caused
a complete or partial collapse in the mobility of 11 novel Cdk1
substrates in addition to Slk19, arguing that phosphorylation of
these proteins in vivo is Cdk1-dependent (Fig. 3c).
We also analysed two previously identified Cdk1 substrates,
Orc6
18
and Pds1
20
. These proteins, as well as some of our candidates,
were only partially dephosphorylated upon inhibitor addition
(Fig. 3c). An underlying assumption in all approaches involving
kinase inhibition in vivo is that phosphates on proteins are rapidly
turned over in the cell. The steady-state phosphate content at any
phosphorylation site is determined by the relative rates of the
protein kinase(s) and protein phosphatase(s) acting on that site.
For some substrates, it is conceivable that kinase activity is present
in 100- or 1,000-fold excess over phosphatase activity, in which case
90 or 99% inhibition of the kinase might not lead to a detectable
drop in the phosphate content of the substrate. Thus, this method
will work only when the relevant phosphatase activity is present in
significant amounts.
Our results suggest that a significant fraction of the 181 candidate
substrates are Cdk1 targets in vivo. Because of limitations in our
analysis, however, it is not yet possible to accurately estimate the
total number of bona fide Cdk1 targets on our list. New methods
will be required to allow more rapid analysis of the phosphorylation
state in vivo of large numbers of proteins in the presence and
absence of Cdk1 activity. Recently, for example, Ficarro et al.
25
used
mass spectrometry to sequence large numbers of phosphopeptides
from yeast extracts. Peptides from three of our 181 best substrates
(Mcm3, Mob1 and YDL113C) were found to be phosphorylated at
Cdk consensus sites in their analysis. Similar studies with lysates of
cdk1-as1 cells, with and without 1-NM-PP1, could provide a global
approach to validating Cdk1 substrate candidates.
About one-third of the 181 best substrates, like those listed in
Table 2, are involved in processes that are regulated during the cell
cycle. Many of these processes are governed by Cdk1, but the
Table 2 Forty selected Cdk1 substrates
Protein* Peak† Full Cdk
consensus sites
Estimated protein
level (ng)‡
Phosphorylation
(arbitrary units)
P-score Biological process
...................................................................................................................................................................................................................................................................................................................................................................
Cdk1 regulation
Cdh1 6 25 304,756 4.1 APC activating factor in M/G1
Cln2 G1 1 30 0 0.0 G1-specific cyclin controlling events at START
Far1 G2/M 4 100 65,166 2.8 Mating-specific Cdk1 inhibitor
Mih1 2 5 35,512 3.9 Phosphatase that dephosphorylates Y19 on Cdk1
Sic1 M/G1 3 200 996,630 3.7 Clb-specific Cdk1 inhibitor
Swe1 G1 2 2 87,408 4.6 Protein kinase that phosphorylates Y19 on Cdk1
Swi5 G2/M 8 0.1 5,206 4.7 G1-specific transcription factor
DNA replication
Cdc6 M/G1 5 0.1 0 0.0 Required for pre-replicative complex formation
Dbf4 2 0.1 5,310 4.7 Regulatory subunit of the protein kinase Cdc7
Eco1 G1 1 30 127,694 3.6 Required for establishment of sister chromatid cohesion
Mcm3 M/G1 5 300 372,637 3.1 Required for initiation of DNA replication
Orc2 6 200 730,607 3.6 Required for pre-replicative complex formation
Orc6 4 50 588,875 4.1 Required for pre-replicative complex formation
Sld2 G1 5 50 51,144 3.0 Required for initiation of DNA replication
Smc4 5 0.1 1,373 4.1 Subunit of the condensin protein complex
Mitosis
Cdc20 G2/M 1 2.5 6,138 3.4 APC-activating factor in anaphase
Cdc5 G2/M 1 5 7,404 3.2 Polo-like kinase, promotes anaphase and mitotic exit
Dbf2 G2/M 2 50 80,255 3.2 Protein kinase required for mitotic exit
Dbf20 S/G2 1 20 31,795 3.2 Protein kinase required for mitotic exit
Fkh2 3 0.1 2,735 4.4 Transcription factor regulating G2/M gene expression
Lte1 8 0.1 3,749 4.6 GDP exchange factor involved in mitotic exit
Mob1 G2/M 2 50 305,565 3.8 Regulatory subunit of Dbf2, required for mitotic exit
Ndd1 G1 4 10 24,327 3.4 Interacts with Fkh2 to contol G2/M gene expression
Net1 3 0.1 4,452 4.6 Regulator of the phosphatase Cdc14
Pds1 G1 3 25 32,213 3.1 Regulator of Esp1 (separase)
Spindle assembly
Ase1 G2/M 7 0.1 555 3.7 Microtubule binding protein required for spindle stability
Kar3 G1 2 75 182,904 3.4 Kinesin-like protein required for proper spindle assembly
Kip2 S/G2 2 150 91,993 2.8 Kinesin-like protein required for proper spindle assembly
Kip3 S/G2 1 50 7,592 2.2 Kinesin-like protein required for proper spindle assembly
Slk19 G1 3 0.1 17,857 5.3 Kinetochore protein required for spindle assembly
Stu2 S 1 50 5,178 2.0 SPB component regulating microtubule dynamics
Actin polarization
Bem1 G2/M 2 100 44,466 2.6 Protein required for proper cell polarization
Bem3 5 0.1 596 3.8 GTPase-activating protein for Rho1 & Cdc42
Bni1 3 0.1 15,168 5.2 Protein involved in Rho protein signal transduction
Other process
Ash1 M/G1 7 5 2,013 2.6 Daughter-cell-localized transcription factor
Bbp1 G1 1 10 13,328 3.1 Spindle-pole-body-associated protein
Chs2 G2/M 4 50 2,744,790 4.7 Chitin synthase II required for proper septum formation
Gin4 G1 2 0.1 781 3.9 Protein kinase required for septin organization
Rad9 9 0.1 604 3.8 DNA damage checkpoint protein
Rts1 2 0.1 2,281 4.4 PP2A regulatory subunit
...................................................................................................................................................................................................................................................................................................................................................................
*Saccharomyces Genome Database name (http://www.yeastgenome.org/). Protein names in bold encode previously identified Cdk1 substrates (see main text).
†Cell-cycle-dependent mRNA transcription peak
8
.
‡In those cases where protein was not detectable, an arbitrary value of 0.1 ng was assigned.
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature862 © 2003 Nature Publishing Group
molecular targets of Cdk1 have remained unidentified. Mitotic
spindle assembly, for example, involves a broad range of molecular
motors and other microtubule-associated proteins, but little is
known about how these components are regulated. Several of
these proteins were identified as Cdk1 substrates in our screen,
and it seems likely that the analysis of their regulation by Cdk1 will
lead to insights into the control of spindle assembly and stability
during mitosis.
The majority of the substrates, including about 50 that are
encoded by ORFs with no known function, have not been impli-
cated previously in cell-cycle-dependent events. Studies of these
substrates may lead to the discovery of unforeseen regulatory
connections in cell-cycle control.
It is now possible to estimate the total number of Cdk1 targets in
the proteome. Of the random 198 proteins we tested, sixteen (8.1%)
had P-scores of 2.0 or greater. If this fraction is representative of the
entire yeast proteome, then there may be about 500 Cdk1 substrates
in yeast. A number of this magnitude does not seem unreasonable,
given that we did not test ,900 ORFs that have a single full Cdk
consensus site and thousands more that contain multiple copies of
the minimal consensus site. Identification of all Cdk1 substrates will
require scanning the entire proteome, either by automated methods
with proteomic libraries or by the identification of endogenous
Cdk1-as1 targets in cell lysates.
It is possible to generate analogue-sensitive mutant forms of
almost any protein kinase, although in some cases multiple
mutations are required to produce a mutant that retains normal
enzymatic activity
26,27
. Our method can therefore be used for the
systematic identification of substrates for any protein kinase. Such
comprehensive analyses of protein kinase targets are clearly an
important step towards extracting useful functional information
from genome sequence. A
Methods
Yeast methods
All strains were derivatives of W303 or S288C, and were grown at 30 8C unless otherwise
noted. Construction of epitope-tagged strains was performed as described
28
. Ase1 and
Slk19 were Myc
13
-tagged, Nbp1 was TAP-tagged, and Fin1, Orc6 and Pds1 were HA
3
-
tagged. C-terminally TAP-tagged versions of 169 of the 181 candidate substrates, expressed
at their endogenous loci, were obtained from a proteomic library kindly provided by
E. K. O’Shea and J. S. Weissman. For western blotting, protein extracts were prepared by
bead beating in urea lysis buffer (20 mM Tris-HCl pH 7.4, 7 M urea, 2 M thiourea, 4%
CHAPS, 1% DTT, 50mM NaF, 80 mM beta-glycero-phosphate, 1 mM sodium
orthovanadate, 1 mM PMSF). Immunoprecipitation and phosphatase assays were
performed as described
13
.
Kinase assays in cell extracts
Cdk1-His
6
, Cdk1-as1-His
6
, and MBP-Clb2 were purified as described
6
. Active Cdk1-
as1zClb2-TAP was purified from cdk1-as1 sic1
D
pGAL-CLB2-TAP yeast cells as described
29
,
except that cell lysates were prepared by bead beating. GST-tagged protein expression was
performed as described
7
. Spheroplasting was performed using Zymolyase-100T
(Seikagaku America Inc.) in 1.5-ml deep-well 96-well plates. Cells were disrupted by
hypotonic lysis with detergent (25 mM Hepes-NaOH pH 7.5, 10mM NaCl, 1 mM EDTA,
0.1% Triton X-100, 1 mM PMSF, 1
m
gml
21
aprotinin, leupeptin, pepstatin A) and
proteins were solubilized by the addition of 300 mM NaCl for 10 min (resulting in efficient
extraction of nuclear proteins). Phosphatase inhibitors were not present during lysate
preparation, which generally results in dephosphorylation of proteins in the lysate. Lysates
were clarified by centrifugation. 500
m
g of whole-cell extract was mixed with 7 nM
Cdk1-as1zMBP-Clb2 or 7 nM Cdk1-as1zClb2-TAP, a creatine-kinase-based 1 mM
ATP-regenerating system, 10 £kinase reaction buffer (200 mM Hepes-NaOH pH 7.4,
100 mM NaCl, 10 mM MgCl
2
), 10 £phosphatase inhibitors (500 mM NaF, 800 mM
b
-glycerophosphate, 10 mM Na
3
VO
4
), and 5
m
Ci
g
-
32
P-N
6
-(benzyl) ATP
30
and brought to
a final volume of 200
m
l. After a 30-min reaction at room temperature, GST-tagged
proteins were purified as described previously
7
. Glutathione eluates were loaded onto 9%
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) gels, silver-
stained, and exposed to a PhosphorImager cassette (Molecular Dynamics) for 72 h.
A key advantage of our approach is that the kinase reaction is performed in a whole-cell
extract, which approximates the normal cellular environment better than typical kinase
reactions performed with purified substrates. Protein kinases in crude extracts are exposed
to a wide variety of potential protein substrates. If kinase and substrate concentrations are
minimized, as in our studies, then only targets with high affinity for the kinase will be
phosphorylated, while non-specific phosphorylation events will be reduced.
In the absence of the ATP-regenerating system, the addition of radiolabelled
N
6
-(benzyl) ATP leads to the phosphorylation of several proteins in the extract,
indicating that some protein kinases are able to use the bulky analogue at a low but
appreciable rate (data not shown). This background phosphorylation is presumably lost in
the presence of high ATP concentrations (Fig. 1, lane 1) because wild-type kinases are
more selective for regular ATP. By contrast, the Cdk1-as1 mutant is much more selective
for the radiolabelled bulky analogue, so that its activity with the analogue remains high
even in the presence of abundant ATP. It is likely that the addition of ATP also protects the
radiolabelled ATP analogue from hydrolysis by the abundant ATPases in the cell extract.
Quantification of phosphorylation, protein levels and P-score
For each GST-tagged protein, phosphorylation levels were measured using Imagequant
(Molecular Dynamics). Many of the tagged proteins were accompanied by multiple
degradation products, which were present before cell lysis, as they were present even when
cells were lysed in boiling SDS–PAGE sample buffer (data not shown). For proteins with
multiple degradation products, the phosphorylation level represents the sum of the
phosphorylation of every degradation product. Phosphorylation levels were normalized
between gels and radiolabelled analogue batches by measuring background radioactivity
levels for each gel. Protein levels were estimated by comparing protein band intensity, size
and shape to BSA standards. P-scores were calculated by taking thelogarith micvalue of the
normalized phosphorylation divided by the estimated protein level.
For simplicity, we assumed in these measurements that all silver-stained protein bands
beneath the full-length tagged protein are fragments of that protein. However, some of
these bands may represent associated proteins. It is remotely possible that these proteins
were phosphorylated, resulting in an artefactual increase in the apparent P-score.
Similarly, fragmentation of tagged proteins could lead to false positives due to exposure of
phosphorylation sites that are normally inaccessible.
Wesuspect that several proteins were not phosphorylated in our screen but are actually
Cdk1 substrates. One possible cause of false negatives is that degradation of tagged
proteins can result in the loss of phosphorylation sites. Cln2, for example, was detected
only as short N-terminal fragments lacking the C-terminal region that contains the Cdk
consensus sites. Another source of false negatives is that many of the tagged proteins are
Figure 3 Validation of yeast Cdk1 substrates in vivo.a, Slk19 is a phosphoprotein with
maximal phosphorylation in mitosis. Slk19-Myc
13
was immunoprecipitated from a lysate
of asynchronous cells (A) or cells arrested in G1 with alpha factor (
a
F), in S phase with
hydroxyurea (HU), or in mitosis with nocodazole (Noc), and immunoblotted with anti-Myc
antibodies. Immunoprecipitates in the last two lanes were treated with lambda
phosphatase, with or without phosphatase inhibitors. Asterisk indicates separase
cleavage products. b, Inhibition of Cdk1 results in the rapid dephosphorylation of Slk19.
Asynchronous cdk1-as1 SLK19-MYC
13
cells were treated with DMSO or 5
m
M 1-NM-PP1
for the indicated time, lysed in urea buffer, analysed by SDS–PAGE, and immunoblotted
for Slk19-Myc
13
, Clb2 and Cdk1. c, Cdk1 inactivation causes a complete or partial
collapse in the mobility of 12 new Cdk1 substrates. Asynchronous yeast cultures
containing the indicated epitope-tagged protein were grown to mid-log phase, treated
with DMSO (2)or20
m
M 1-NM-PP1 (þ) for 15 min at 30 8C, lysed in urea lysis buffer,
and analysed by immunoblotting.
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature 863
© 2003 Nature Publishing Group
a A aF
HU
Noc Noc Noc
Slk19{I
Slk19*{,_
________
__.
+ +
11.
phosphatase
+
11.
phosphatase inhibitor
b DMSO 5
µM
1-
NM
-
PP1
Time
(min)
O 5 10 15
30
60 0 5 10 15
30
60
Slk19 { •
Slk19*{
_____
_
Clb2
-----
__
_
Cdk1
---....._.
___
,__
__
__
__.
_.
C
Ase1
Dpb2
Fin1
Fun30 Mlh2
Nbp1
Orc6
B ~ e:;JE:;;J~
~~
1-NM-PP1 -+ - + + + - + + - +
Pds1
Sen1
Sfb3 Slk19 Swi6 Ykr089c Ypr174c
c:i
~~
~,---1
--,11
-
-11--1
1-
NM
-
PP1
-+ - + - + - + - + + +
not present in cell lysates in appreciable amounts. Cdc6, for example, was not detectable in
cell lysates by silver stain or western blot, suggesting that it is either not expressed in the
library or that it is insoluble in our lysis conditions. Because of the method by which the
GST-ORF library was constructed, it is likely that each yeast strain contains a mixture of
cells expressing either GST alone or the desired GST-tagged protein. As there is no
selection for the GST-ORF-containing cells, long-term culture could result in cell lysates
containing very low levels of GST-tagged protein and high levels of GST (as illustrated in
Fig. 2a, bottom panel). This may explain the very low levels of tagged protein expression
we observed (Fig. 2c), and suggests that there are probably more Cdk substrates among the
156 proteins that were not detectable by silver stain and apparently not phosphorylated.
Received 11 March; accepted 19 September 2003; doi:10.1038/nature02062.
1. Morgan, D. O. Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell Dev.
Biol. 13, 261–291 (1997).
2. Nigg, E. A. Mitotic kinases as regulators of cell division and its checkpoints. NatureRe v.Mol. Cell Biol.
2, 21–32 (2001).
3. Shah, K., Liu, Y., Deirmengian, C. & Shokat, K. M. Engineering unnatural nucleotide specificity for
Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates. Proc. Natl Acad. Sci. USA 94,
3565–3570 (1997).
4. Shah, K. & Shokat, K. M. A chemicalgenetic screen for direct v-Src substrates reveals ordered assembly
of a retrograde signaling pathway. Chem. Biol. 9, 35–47 (2002).
5. Witucki, L. A. et al. Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure
and phospho-acceptor specificity of the wild-type enzyme. Chem. Biol. 9, 25–33 (2002).
6. Bishop, A. C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407,
395–401 (2000).
7. Martzen, M. R. et al. A biochemical genomics approach for identifying genes by the activity of their
products. Science 286, 1153–1155 (1999).
8. Spellman, P. T. et al. Comprehensive identification of cell cycle-regulated genes of the yeast
Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273–3297 (1998).
9. Moll, T., Tebb, G., Surana, U., Robitsch, H. & Nasmyth, K. The role of phosphorylation and the
CDC28 protein kinase in the cell cycle-regulated nuclear import of the S. cerevisiae transcription
factor SWI5. Cell 66, 743–758 (1991).
10. Verma, R. et al. Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S
phase. Science 278, 455–460 (1997).
11. Deshaies, R., Chau, V. & Kirschner,M. Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent
pathway. EMBO J. 14, 303–312 (1995).
12. Zachariae, W., Schwab, M., Nasmyth, K. & Seufert, W. Control of cyclin ubiquitination by CDK-
regulated binding of Hct1 to the Anaphase Promoting Complex. Science 282, 1721–1724 (1998).
13. Jaspersen, S. L., Charles, J. F. & Morgan, D.O. Inhibitory phosphor ylation of the APC regulatorHct1
is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr. Biol. 9, 227–236 (1999).
14. Gartner, A. et al. Pheromone-dependent G1 cell cycle arrest requires Far1 phosphorylation, but may
not involve inhibition of Cdc28-Cln2 kinase, in vivo.Mol. Cell. Biol. 18, 3681–3691 (1998).
15. Mortensen, E. M., McDonald, H., Yates, J. III & Kellogg, D. R. Cell cycle-dependent assembly of a
Gin4-septin complex. Mol. Biol. Cell 13, 2091–2105 (2002).
16. McMillan, J. N., Theesfeld, C. L., Harrison, J. C., Bardes, E. S. & Lew, D. J. Determinants of Swe1p
degradation in Saccharomyces cerevisiae.Mol. Biol. Cell 13, 3560–3575 (2002).
17. Elsasser, S., Chi, Y., Yang, P.& Campbell, J. L. Phosphorylation controls timing of Cdc6p destruction:
A biochemical analysis. Mol. Biol. Cell 10, 3263–3277 (1999).
18. Nguyen, V. Q., Co, C. & Li, J. J. Cyclin-dependent kinases prevent DNA re-replication through
multiple mechanisms. Nature 411, 1068–1073 (2001).
19. Masumoto, H., Muramatsu, S., Kamimura, Y. & Araki, H. S-Cdk-dependent phosphorylation of Sld2
essential for chromosomal DNA replication in budding yeast. Nature 415, 651–655 (2002).
20. Agarwal, R. & Cohen-Fix, O. Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is
required for efficient nuclear localization of Esp1/separase. Genes Dev. 16, 1371–1382 (2002).
21. Rudner, A. D. & Murray, A. W. Phosphorylation by Cdc28 activates theCdc20-dependent activ ityof
the anaphase-promoting complex. J. Cell Biol. 149, 1377–1390 (2000).
22. Roberts, J. M. Evolving ideas about cyclins. Cell 98, 129–132 (1999).
23. Cross, F. R., Yuste-Rojas,M., Gray, S. & Jacobson, M. D. Specialization and targeting of B-type cyclins.
Mol. Cell 4, 11–19 (1999).
24. Sullivan, M., Lehane, C. & Uhlmann, F. Orchestrating anaphase and mitotic exit: separase cleavage
and localization of Slk19. Nature Cell Biol. 3, 771–777 (2001).
25. Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its application to
Saccharomyces cerevisiae.Nature Biotechnol. 20, 301–305 (2002).
26. Niswender, C. M. et al. Protein engineering of protein kinase A catalytic subunits results in the
acquisition of novel inhibitor sensitivity. J. Biol. Chem. 277, 28916–28922 (2002).
27. Habelhah, H. et al. Identification of new JNK substrate using ATP pocket mutant JNK and a
corresponding ATP analogue. J. Biol. Chem. 276, 18090–18095 (2001).
28. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and
modification in Saccharomyces cerevisiae.Ye as t 14, 953–961 (1998).
29. Puig, O.et al. The tandem affinity purification (TAP)method: a general procedure of protein complex
purification. Methods 24, 218–229 (2001).
30. Kraybill, B. C., Elkin, L. L., Blethrow, J. D.,Morgan, D. O. & Shokat, K. M. Inhibitor scaffolds as new
allele specific kinase substrates. J. Am. Chem. Soc. 124, 12118–12128 (2002).
Supplementary Information accompanies the paper on www.nature.com/nature.
Acknowledgements We thank E. Phizicky for the GST-ORF library; the many laboratories at
UCSF who helped duplicate and distribute the GST-ORF library; E. K. O’Shea and J.S. Weissman
for TAP-tagged strains; D. Kellogg for sharing unpublished results; E. K. O’Shea and R. Deshaies
for expression plasmids; N. Dephoure and M. Verzifor strain construction; S. Biggins, D. Kellogg,
A. Goga, S. Steggerda, C. Carroll, D. Randle, M. Enquist-Newman and M. Loog for advice and
critical reading of the manuscript. This work was supported by funding from the National
Institute of General Medical Sciences (D.O.M.),the National Institutes of Health (K.M.S.), and by
a predoctoral fellowship from the National Science Foundation (J.A.U.).
Competing interests statement The authors declare that they have no competing financial
interests.
Correspondence and requests for materials should be addressed to D.O.M.
(dmorgan@cgl.ucsf.edu).
letters to nature
NATURE| VOL 425 | 23 OCTOBER 2003 | www.nature.com/nature864 © 2003 Nature Publishing Group
