Tài liệu Báo cáo khoa học: Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases pptx

Phosphorylation of cyclin dependent kinase 4 on
tyrosine 17 is mediated by Src family kinases
Nicholas G. Martin, Peter C. McAndrew, Paul D. Eve and Michelle D. Garrett
Cancer Research UK Centre for Cancer Therapeutics at The Institute of Cancer Research, Haddow Laboratories, Sutton, UK
Cyclin dependent kinase (CDK) 4 ⁄ cyclin D kinase is
an important regulator of cell cycle entry and G1 pro-
gression, where it initiates inhibitory phosphorylation
of the retinoblastoma tumour suppressor protein RB
[1–3], a critical step for progression into the S-phase
[4–6]. Unsurprisingly, considering its pivotal role in cell
cycle control, CDK4 ⁄cyclin D kinase activity is com-
monly deregulated in cancer. The majority of cancers
contain at least one genetic alteration that affects the
RB pathway [7], and a better understanding of how
CDK4 ⁄ cyclin D kinase is controlled could provide new
therapeutic targets and strategies for the treatment of
cancer.
The activity of the CDK4 ⁄ cyclin D holoenzyme is
regulated by multiple mechanisms, the most important
of which is the association of CDK4 with the D-type
cyclin subunit, a requirement for kinase activity [8,9].
One of the most poorly understood mechanisms by
which CDK4 ⁄ cyclin D kinase activity is controlled is
phosphorylation at tyrosine 17 (Y17) of CDK4. This
site corresponds to tyrosine 15 (Y15) of CDK1 (Cdc2),
a site of inhibitory phosphorylation on this kinase
[10,11]. Phosphorylation on Y17 of CDK4 has been
shown to occur in mammalian cells that are entering
the cell cycle from quiescence, and then undergo G1
arrest induced by UV irradiation [12,13]. In these stud-
ies, the activation of wild-type CDK4 was inhibited by

UV irradiation, whereas a Y17F nonphosphorylatable
mutant form of CDK4 was activated normally, dem-
onstrating that phosphorylation of CDK4 on Y17 is
inhibitory to kinase activity [12]. Expression of the
Y17F mutant of CDK4 abrogated the UV-induced G1
Keywords
CDK4; C-YES; Src; tyrosine phosphorylation;
WEE1
Correspondence
M. D. Garrett, Cancer Research UK Centre
for Cancer Therapeutics at The Institute of
Cancer Research, Haddow Laboratories, 15
Cotswold Road, Sutton, Surrey SM2 5NG,
UK
Fax: +44 020 87224126
Tel: +44 020 87224352
E-mail:
(Received 19 December 2007, revised 19
March 2008, accepted 11 April 2008)
doi:10.1111/j.1742-4658.2008.06463.x
Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activ-
ity is frequently deregulated in cancer. The activity of cyclin dependent
kinase 4 is controlled by multiple mechanisms, including phosphorylation
of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent
kinase 1, which undergoes inhibitory phosphorylation by WEE1 and
MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4
on tyrosine 17 are still unknown. In the present study, we generated a
phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin
dependent kinase 4, and showed that this site is phosphorylated to a low
level in asynchronously proliferating HCT116 cells. We purified tyrosine 17

kinases from HeLa cells and found that the Src family non-receptor tyro-
sine kinase C-YES contributes a large fraction of the tyrosine 17 kinase
activity in HeLa lysates. C-YES also phosphorylated cyclin dependent
kinase 4 when transfected into HCT116 cells, and treatment of cells with
Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of
cyclin dependent kinase 4. Taken together, the results obtained in the
present study provide the first evidence that Src family kinases, but not
WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17,
and help to resolve how the phosphorylation of this site is regulated.
Abbreviations
CDK, cyclin dependent kinase; TGF, transforming growth factor; Y15, tyrosine 15; Y17, tyrosine 17.
FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3099
cell cycle arrest, leading to an increase in chromosomal
aberrations and cell death, indicating that phosphory-
lation of this site is important for integrity of the G1
checkpoint [13]. Phosphorylation of CDK4 on Y17
has also been detected in cells entering quiescence in
response to contact inhibition, serum starvation and
treatment with transforming growth factor (TGF) b
[12,14]. Taken together, these studies suggest that Y17
phosphorylation of CDK4 regulates G1 phase cell
cycle arrest and quiescence.
The available evidence suggests that the dual speci-
ficity phosphatase CDC25A controls removal of phos-
phate from Y17 of CDK4. The increase in Y17
phosphorylation brought about by TGFb corresponds
to a loss of CDC25A [14], and an increase in Y17
phosphorylation of CDK4 has been detected upon
chemical inhibition of CDC25A [15–17]. Although
CDC25A may dephosphorylate the CDK4 Y17 site,

the kinase(s) that phosphorylate this residue remain
unknown. The obvious candidate kinases for this role
are WEE1 and MYT1 because they phosphorylate
CDK1 on Y15; however, neither is able to phosphory-
late CDK4 on Y17 in vitro [18,19]. In the present
study, we used column chromatography to purify
CDK4 Y17 kinases from HeLa cell extracts, and found
that the cellular phosphorylation of CDK4 on Y17 is
mediated by Src family kinases.
Results
Detection of CDK4 Y17 phosphorylation and
kinase activity
The N-termini of CDKs 1, 2, 4 and 6 are highly con-
served and there is an equivalent residue to the Y17 site
of CDK4 in each of these kinases. To allow the study of
CDK4 Y17 phosphorylation, a phosphospecific anti-
body to the Y17 site was raised using a 13mer phospho-
peptide as the immunogen (Fig. 1A). The antibody was
purified and was found to be highly specific for the
phosphopeptide over the nonphosphopeptide by ELISA
(data not shown). To test the site and phosphospecificity
of the antibody against full length CDK4, Flag-tagged
CDK4 or the nonphosphorylatable Y17F mutant of
CDK4 were transfected into HCT116 cells. Western
blotting using a CDK4 antibody confirmed expression
of the exogenous Flag-CDK4 and Flag-CDK4
Y17F
because they migrate more slowly on the SDS ⁄ PAGE
gel than the endogenous CDK4 due to the Flag-tag
(Fig. 1B). Western blotting of Flag-immunoprecipitates

from these cell lysates with the CDK4 Y17 phospho-
specific antibody (CDK4 pY17) revealed a low basal
level of Y17 phosphorylation that was greatly
induced by treatment of the cells with the protein
tyrosine phosphatase inhibitor sodium orthovanadate.
No signal was detected in the Flag-CDK4
Y17F
immuno-
precipitates, indicating that the antibody is both
phosphospecific and site specific. The low basal signal of
CDK4 Y17 phosphorylation is in keeping with
previous reports that did not detect CDK4 Y17
phosphorylation in asynchronously proliferating cells
[12,13].
A
B
C
Fig. 1. Detection of CDK4 Y17 phosphorylation. (A) Alignment of
the N-terminal amino acids of CDKs 1, 2, 4 and 6 showing the con-
served tyrosine residue (vertical box) that corresponds to Y17 of
CDK4, and the peptide used to raise the pY17 antibody (horizontal
box). (B) HCT116 cells were transfected with Flag-tagged CDK4 or
CDK4
Y17F
and were treated with or without sodium orthovanadate
(200 l
M). Lysates were immunoprecipitated with the Flag antibody
(Flag IP). The immunoprecipitates and lysates were western blotted
with the phosphospecific CDK4 pY17, total CDK4 and aTubulin anti-
bodies as indicated. Long and short exposures of the CDK4 pY17

blot are shown. (C) Lysates from HT29, HCT116, HeLa cells and
HeLa nuclei were assayed for Y17 kinase activity using the 96-well
plate format assay. Each sample contained 2.5 lg of total protein.
Values are the average of four samples with error bars indicating
the SEM.
Phosphorylation of CDK4 by Src family kinases N. G. Martin et al.
3100 FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS
Using the CDK4 pY17 antibody, we developed a
96-well plate format kinase assay to detect Y17 kinases.
The 13mer non-phosphopeptide was used as the
substrate for the kinase(s), and phosphorylation of the
peptide was detected using the CDK4 pY17 antibody
and a Europium labelled secondary antibody. Cell
lysates from a range of cell lines were tested for CDK4
Y17 kinase activity (Fig. 1C), and activity was detected
in all cell lysates with no activity in the buffer control.
Interestingly, a higher level of activity was detected in
HeLa nuclear lysate compared to HeLa whole cell
lysate. Due to the commercial availability of suitable
quantities of HeLa nuclear pellets, these were chosen as
the starting material for a purification of CDK4 Y17
kinases.
Purification of C-YES as a CDK4 Y17 kinase
A five-step procedure was used to purify Y17 kinases
from HeLa nuclei (Table 1). After each purification
step, the Y17 kinase activities and the protein concen-
trations of the resulting fractions were measured, and
a final purification of approximately 939-fold was
achieved. Purification by butyl-sepharose chromato-
graphy resolved the majority of the Y17 kinase activity

as a large peak between fractions 96 and 106, towards
the end of the ammonium sulfate gradient (Fig. 2A).
By contrast, western blotting of selected fractions with
a WEE1 antibody revealed that WEE1 flowed through
the column without binding to the butyl-sepharose
resin (Fig. 2B). No Y17 kinase activity was detected in
the corresponding fractions (Fig. 2A), consistent with
the previous reports showing that WEE1 does not
phosphorylate CDK4 on Y17 [19].
The Y17 kinase activity eluted from the hydroxyapa-
tite column as a single peak between fractions 40 and
47, at the start of the phosphate gradient (Fig. 2C).
Fractions 38–49 and a sample of the input were precip-
itated using deoxycholate and trichloroacetic acid, and
the proteins were separated using SDS ⁄ PAGE. A
major protein band of approximately 60 kDa was
detected by staining with coomassie (Fig. 2D). This
band tracked with CDK4 Y17 kinase activity with a
peak in fraction 42 (Fig. 2C). A sample of the band
was excised and the constituent proteins were analysed
by Q-TRAP MS (Applied Biosystems, Foster City,
CA, USA). Forty-nine ions were selected from the
sample and sequenced by MS ⁄ MS. Forty-seven of
these were peptides from the chaperonin HSP60 and
two were peptides from the nonreceptor tyrosine
kinase C-YES. Selected fractions from each of the
chromatography steps were western blotted with an
antibody specific for C-YES, and C-YES protein
tracked with CDK4 Y17 activity over all of the
chromatography columns (Fig. 2B,E).

Src family kinases but not WEE1 or MYT1
phosphorylate CDK4 on Y17 in vitro
To confirm that C-YES contributes to the Y17 kinase
activity found in cell lysates, C-YES was immunode-
pleted from HeLa nuclear and whole cell extracts, and
both the supernatants and the precipitates were
assayed for Y17 kinase activity. C-YES was success-
fully immunodepleted from the nuclear and whole cell
lysates and C-YES protein appeared in the precipitates
(Fig. 3A). Mock depletions where the depleting anti-
body was substituted for buffer were used as negative
controls. The supernatants were assayed for Y17
kinase activity using both the tube (Fig. 3A) and
96-well plate (Fig. 3B) format assays, whereas the pre-
cipitates were assayed using the tube format assay
only. Depletion of C-YES from both the nuclear and
whole cell lysates resulted in a concomitant reduction
in kinase activity as measured by both plate and tube
assays. The accumulation of C-YES in the precipitates
correlated with the appearance of kinase activity in
those samples. The level of depletion of kinase activity
mirrored the level of depletion of C-YES, indicating
that C-YES contributes a large fraction of the Y17
kinase activity in these lysates.
Immunodepletion from lysates was repeated for the
CDK1 Y15 kinases WEE1 (Fig. 3C,E) and MYT1
(Fig. 3D,E). In both cases, the proteins were
Table 1. CDK4 Y17 kinase activity purification from HeLa nuclei.
Purification step
Activity total

(counts)
Protein
(mg)
Specific activity
(countsÆmg
)1
)
Recovery
(%)
Purification
(fold)
Nuclear lysate 1.4 · 10
11
1670 8.4 · 10
7
100 1
(NH
4
)
2
SO
4
precipitation 2.9 · 10
11
596 4.8 · 10
8
205 5.7
Butyl-sepharose 8.5 · 10
10
16.2 5.3 · 10

9
61 62.7
Q-sepharose 9.6 · 10
9
1.24 7.4 · 10
9
6.8 88.8
Superdex 200 1.0 · 10
10
0.51 2.0 · 10
10
7.2 237
Hydroxyapatite 7.3 · 10
9
0.09 7.9 · 10
10
5.2 939
N. G. Martin et al. Phosphorylation of CDK4 by Src family kinases
FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3101
successfully depleted from HeLa whole cell lysates;
however, this did not result in a loss of Y17 kinase
activity. In addition, the WEE1 and MYT1 immuno-
precipitates did not possess Y17 kinase activity. These
data indicate that WEE1 and MYT1 do not contribute
to the CDK4 Y17 kinase activity in these lysates and
are unlikely to be CDK4 kinases in cells.
As further confirmation that the Y17 kinase in HeLa
whole cell lysates is a Src family kinase, we assayed its
sensitivity to the Src family kinase inhibitor PP2 [20].
In comparison, we also assayed the inhibition of puri-

fied recombinant kinases C-SRC, C-YES and p49
WEE1
by PP2 using the 96-well plate format kinase assay.
p49
WEE1
is a truncated form of human WEE1 that is
known to have altered substrate specificity with respect
to full length WEE1 [18,21] and has CDK4 Y17 kinase
activity in vitro (Fig. 4A). PP2 had similar potency
against C-SRC, C-YES and the HeLa lysate, but did
not inhibit p49
WEE1
activity at any concentration. This
confirms that the Y17 kinase found in HeLa lysate is
PP2 sensitive and likely to be a Src family kinase.
To test whether CDK4 can be a substrate for Src
family kinases other than C-YES, recombinant, puri-
fied C-SRC, C-YES and LYN were assayed for Y17
kinase activity using full length CDK4 in a tube
format kinase assay (Fig. 4B). All three kinases phos-
phorylated the Y17 site, demonstrating that CDK4
kinase activity is not restricted to C-YES in vitro.
Src family kinases phosphorylate CDK4 on Y17
in cells
To determine whether C-YES can phosphorylate
cellular CDK4, empty vector, C-YES, hyperactive
C-YES
Y537F
or kinase dead C-YES
K305R

were cotrans-
fected with empty vector, Flag-CDK4 or Flag-
CDK4
Y17F
into HCT116 cells (Fig. 5A,B). The Y537
residue of C-YES is equivalent to Y527 of C-SRC and
is the site of inhibitory phosphorylation by the pro-
tein-tyrosine kinase CSK [22]. The exogenous C-YES,
C-YES
Y537F
and C-YES
K305R
were detected in the cell
lysates by western blotting with a C-YES antibody,
and appeared as a double band that migrated more
slowly than endogenous C-YES due to the C-terminal
Myc-His-tag. The exogenous CDK4 proteins were
immunoprecipitated with the Flag antibody and Y17
phosphorylation was detected with the CDK4 pY17
antibody. Co-transfection of CDK4 with C-YES
enhanced the level of Y17 phosphorylation compared
to vector alone, and the level was even greater when
the hyperactive C-YES
Y537F
was expressed. This con-
firms that C-YES can regulate the phosphorylation of
B
A
C
D

E
Fig. 2. Identification of C-YES as a Y17 kinase. (A) The ammo-
nium sulfate precipitated HeLa nuclear lysate was fractionated by
butyl-sepharose chromatography and the even numbered frac-
tions were assayed for Y17 kinase activity and protein content.
(B) Selected fractions from the flow-through (16), wash (48) and
gradient (80–116), along with a sample of the column input and
the HeLa nuclear lysate, were separated on a 4–12% NuPAGE
gel and western blotted with WEE1 and C-YES antibodies.
(C) The elute from the superdex 200 gel filtration column was
fractionated by hydroxyapatite chromatography and the fractions
were assayed for Y17 kinase activity and protein content.
Fractions from the gradient and a sample of the column input
were separated on 4–12% NuPAGE gels and proteins were
stained with coomassie (D) and western blotted with the C-YES
antibody (E).
Phosphorylation of CDK4 by Src family kinases N. G. Martin et al.
3102 FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS
CDK4 on Y17 in cells. The kinase dead version of
C-YES did not increase the level of Y17 phosphoryla-
tion (Fig. 5B), confirming that the phosphorylation of
CDK4 is dependent on the kinase activity of C-YES.
To determine whether the Src family kinases are
responsible for the CDK4 Y17 phosphorylation
detected in cells, Flag-CDK4 and Flag-CDK4
Y17F
were transfected into HCT116 cells, which were then
treated with either the vehicle (dimethylsulfoxide) or
the Src inhibitors PP2 and SU6656 [23] (Fig. 5C). Wes-
tern blotting of Flag immunoprecipitates from these

cells with the CDK4 pY17 antibody revealed that both
of the Src inhibitors blocked phosphorylation of
CDK4 on Y17. Interestingly, blotting of the lysates
with a CDK1 pY15 phosphospecific antibody revealed
that the inhibitors did not block the phosphorylation
of CDK1 on this site.
Discussion
The aim of the present study was to identify the kinase
or kinases that phosphorylate CDK4 on Y17, the
equivalent site to Y15 on CDK1. We purified CDK4
Y17 kinase activity from HeLa cells and identified
C-YES as a kinase that contributes a large fraction of
the Y17 kinase activity found in HeLa lysates. C-YES
is a 62 kDa nonreceptor tyrosine kinase of the Src
family that is expressed in a wide range of tissues [24].
The N-terminus of C-YES is dually myristoylated and
palmitoylated [25], and these modifications target
C-YES to intracellular membranes and exclude it from
the nucleus [26]. Considering that C-YES is not usually
localized to the nucleus, it is interesting that we found
a higher concentration of C-YES protein and Y17
kinase activity in our nuclear lysates compared to
A
B
C
D
E
Fig. 3. C-YES, but not WEE1 or MYT1, contributes Y17 kinase activity to HeLa lysates. (A) C-YES was immunodepleted from HeLa whole
cell and nuclear lysates with the C-YES polyclonal antibody and protein G sepharose, and the depleted supernatants and precipitates were
western blotted with the C-YES antibody. The supernatants and precipitates were assayed for Y17 kinase activity using the tube format

kinase assay and western blotting of samples with the phosphospecific CDK4 pY17 antibody and the total CDK4 antibody. As negative
controls, the samples were mock depleted by substitution of the antibody for buffer. The negative controls for the kinase assay were either
no lysate (far left hand lane) or antibody and protein G but no lysate (far right hand lane). *Background caused by cross-reactivity with the
depleting antibody. (B) The C-YES depleted and mock depleted supernatants were assayed for Y17 kinase activity using the 96-well plate
format assay. Each sample contained 2.5 lg of total protein. Values are the average of four samples with error bars indicating the SEM.
HeLa whole cell lysates were depleted with WEE1 and MYT1 antibodies as described for C-YES and the deleted supernatants were assayed
for Y17 kinase activity using the tube format kinase assay (C, D) and the 96-plate format assay (E).
N. G. Martin et al. Phosphorylation of CDK4 by Src family kinases
FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3103
whole cell lysates (Fig. 3A,B). Previous immunofluo-
rescence studies have shown C-YES to be located
mainly at the plasma membrane and perinuclear region
[26]. It is possible that the C-YES found in our nuclear
pellets was not genuinely nuclear and was from perinu-
clear material or other C-YES containing membranes
that were not removed during the nuclear fraction-
ation; however, the potential existence of nuclear
C-YES warrants further investigation.
The study then demonstrated that C-YES phospho-
rylates CDK4 on Y17 when expressed along with
CDK4 in HCT116 cells, indicating that C-YES is a
CDK4 Y17 kinase in cells. Moreover, the level of Y17
phosphorylation was dependent on the activity of
C-YES because the activated C-YES
Y537F
mutant
phosphorylated CDK4 to a higher degree than wild-
type C-YES, and the kinase dead C-YES
K305R
did not

phosphorylate CDK4. We also showed that two struc-
turally unrelated Src family kinase inhibitors block the
phosphorylation of CDK4 on Y17. As these inhibitors
have similar activity against all Src family kinases
[20,23], and we have shown that C-YES, C-SRC and
LYN can all phosphorylate CDK4 on Y17 in vitro,it
is possible that Src kinases other than C-YES are also
involved in the cellular phosphorylation of CDK4. The
A
B
Fig. 4. Src family kinases phosphorylate CDK4 on Y17 in vitro.
(A) HeLa lysates (2.5 lg protein per well), and recombinant purified
p49
WEE1
, C-YES and C-SRC were assayed for Y17 kinase activity
using the plate format assay at various concentrations of the Src
family kinase inhibitor PP2. Values are the average of four samples
normalized to the start activity, with error bars indicating the SEM
converted to a percentage as described in the Experimental proce-
dures. (B) Recombinant purified Src family kinases C-YES, C-SRC
and LYN were assayed for Y17 kinase activity using the tube
format assay and western blotting of the samples with the phos-
phospecific CDK4 pY17 antibody and the total CDK4 antibody.
As negative controls, the kinases was heat inactivated at 95 °C
for 5 min prior to the assay, as indicated.
A
B
C
Fig. 5. Src family kinases phosphorylate CDK4 on Y17 in cells.
HCT116 cells were transfected with Flag-tagged CDK4 or CDK4

Y17F
along with C-YES, activated C-YES
Y537F
(A) or kinase dead
C-YES
K305R
(B) and then treated with or without sodium orthovana-
date as indicated. Lysates were immunoprecipitated with the Flag
antibody (Flag IP). The immunoprecipitates and lysates were wes-
tern blotted with the phosphospecific CDK4 pY17, total CDK4 and
C-YES antibodies as indicated. (C) HCT116 cells were transfected
with Flag-tagged CDK4 or CDK4
Y17F
and treated with either vehicle
(dimethylsulfoxide), PP2 (10 l
M), SU6656 (10 lM) or sodium ortho-
vanadate (200 l
M). Lysates were immunoprecipitated with the Flag
antibody (Flag IP). The immunoprecipitates and lysates were wes-
tern blotted with the phosphospecific CDK4 pY17 and CDK1 pY15
antibodies, total CDK4, CDK1 and C-YES antibodies as indicated.
Long and short exposures of the CDK4 pY17 blot are shown.
Phosphorylation of CDK4 by Src family kinases N. G. Martin et al.
3104 FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS
specific depletion of C-YES from cells using siRNA
did not reduce the level of Y17 phosphorylation (data
not shown); however, this result could be due to inade-
quate knockdown of C-YES or compensation by other
Src family members. It is known that Src family
kinases share many substrates and show considerable

functional redundancy [27], and it is likely the Src
kinases other than C-YES contribute to Y17 phos-
phorylation of CDK4 in cells.
Src family kinases have recently been reported to
phosphorylate p27
KIP1
on two tyrosine residues [28,29]
and there is the possibility that tyrosine phosphoryla-
tion of p27
KIP1
could indirectly affect the Y17 phos-
phorylation of CDK4. However, it is clear that the
CDK4 pY17 antibody does not detect tyrosine phos-
phorylation of p27
KIP1
because a p27
KIP1
band would
run at a lower molecular weight than CDK4 on a wes-
tern blot, and no signal was detected with the
CDK4
Y17F
mutant. Furthermore, the purified recombi-
nant cyclin D1-CDK4 complex used as a substrate for
kinase assays was dimeric and did not contain p27
KIP1
(data not shown). Therefore, the data presented in
Figs 1–4 strongly suggest that Src kinases directly
phosphorylate CDK4 on Y17 and that this phosphory-
lation is independent of p27

KIP1
.
The finding that Y17 phosphorylation is mediated
by Src family kinases is completely novel and it is
interesting to note how this fits in with the previously
published data. CDK4 phosphorylation on Y17 is con-
sidered to restrain cell cycle progression in UV irradi-
ated cells undergoing G0–G1 transit [12,13], and may
play a role in TGFb mediated G1 arrest [14]. In this
context, Y17 phosphorylation is thought to be modu-
lated by loss of the phosphatase CDC25A. Our data
suggest that Src kinases are candidates to provide the
phosphorylation of CDK4. If this is the case, it may
be that Src family kinase activity is important for G1
arrest in these cellular situations or other Y17 depen-
dent processes that are yet to be defined.
It is interesting to note that this is not the first
report of Src family kinases phosphorylating CDKs.
The Src kinase LYN is already known to phosphory-
late CDK1 [30,31] and CDK2 [32] on Y15 in response
to DNA damage, and it is plausible that LYN may
also phosphorylate CDK4 on Y17 in this context. It is
clear, however, that the regulation of CDK4 Y17
phosphorylation differs markedly from the regulation
of CDK1 Y15 phosphorylation. First, the basal level
of CDK4 Y17 phosphorylation appears to be very low
and was greatly increased by incubation with the pro-
tein tyrosine phosphatase inhibitor vanadate (Figs 1B
and 5C). By contrast, the phosphorylation of CDK1
on Y15 was increased to a much lesser degree by vana-

date treatment (Fig. 5C). This is in keeping with previ-
ous studies that have either reported very low or
undetectable levels of Y17 phosphorylation in
untreated asynchronously proliferating cells [12–14,
33–35], and suggests that Y17 phosphorylation of
CDK4 plays little role in an unperturbed cell cycle.
Second, the kinases WEE1 and MYT, which phos-
phorylate CDK1 on Y15, do not appear to phosphory-
late CDK4 on Y17. Immunodepletion of WEE1 or
MYT1 from cell lysates did not deplete Y17 kinase
activity, suggesting that CDK4 is not a substrate for
WEE1 or MYT1, in agreement with previous reports
demonstrating that neither of these kinases phosphory-
late CDK4 in vitro [18,19]. Furthermore, we found
that, although CDK4 phosphorylation on Y17 was
blocked by Src family kinase inhibitors, CDK1 phos-
phorylation on Y15 was not affected (Fig. 5C). This
suggests that Src kinases do not phosphorylate CDK1
during an unperturbed cell cycle.
To conclude, we show that Src family kinases phos-
phorylate CDK4 on Y17 in the cell. Considering the
tight regulation of CDK4 activity during the cell cycle
and the critical role that CDK4 plays in human
cancer, it will be interesting to investigate how this
novel form of regulation affects CDK4 activity during
these processes.
Experimental procedures
Cell lines and cell culture
Frozen whole HeLa cells and HeLa nuclei were purchased
from Cil Biotech (Mons, Belgium). HCT116 and HT29 cells

(ATCC, LGC Promochem, Teddington, UK) were main-
tained in DMEM medium supplemented with 10% (v ⁄ v)
fetal calf serum in an incubator at 37 °C with a humidified
atmosphere of 5% CO
2
. HCT116 cells were transfected
using Effectene reagent in accordance with the manufac-
turer’s instructions (Qiagen, Crawley, UK) and were lysed
or frozen 48 h later.
Purified kinases and inhibitors
Purified recombinant LYN and C-YES were puchased from
Calbiochem (Merck Chemicals Limited, Nottingham, UK),
C-SRC was obtained from Upstate (Lake Placid, NY,
USA) and p49
WEE1
was obtained as previously described
[18]. The kinase inhibitors PP2 and SU6656 were purchased
from Calbiochem (Merck Chemicals Limited) and were dis-
solved at 10 mm in dimethylsulfoxide. The phosphatase
inhibitor sodium orthovanadate (Sigma-Aldrich, Dorset,
UK) was dissolved at 0.1 m in water. Cells were treated
with these inhibitors for 16 h.
N. G. Martin et al. Phosphorylation of CDK4 by Src family kinases
FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3105
Plasmid vectors
The human CDK4 coding sequence was cloned into the
EcoRI-XbaI site of pcDNA3.1 (Invitrogen, Paisley, UK)
modified with a FLAG-tag BamHI-EcoRI. Codon 17 of
CDK4 was mutated from TAT (Tyr) to TTT (Phe) using
the QuickChangeÒ kit (Stratagene, La Jolla, CA, USA)

and the forward primer: 3¢-GAAATTGGTGTCGGCGCC
TTTGGGACAGTGTAC-5¢. The human C-YES coding
sequence minus the termination codon was PCR amplified
from IMAGE clone 5260751 and cloned into the XbaI-NotI
site of pcDNA3.1 ⁄ myc-His A (Invitrogen). Codon 537 of
C-YES was mutated from TAC (Tyr) to TTC (Phe) with
the forward primer: 3¢-GTCACAGAGCCACAGTTCCAG
CCAGGA-5¢. Codon 305 of C-YES was mutated from
AAA (Lys) to AGA (Arg) with the forward primer: 3¢-GG
AACCACGAAAGTAG CAATC AGA ACACTA AAACC A
GGTACAATGATGC-5 ¢ . All vector inserts were
sequenced prior to use.
CDK4 pY17 antibody generation
Murex lop-eared rabbits were injected with a phosphopep-
tide corresponding to amino acids 11–23 of human CDK4
phosphorylated on Y17 with an additional cysteine at the
C-terminus (EIGVApYGTVYKAC) conjugated to Keyhole
Limpet Haemocyanin (Pierce, Rockford, IL, USA). The
resulting antisera was affinity-purified by binding to the
phosphopeptide antigen conjugated to sulfolink media
(Pierce). The antibodies were eluted with 100 mm glycine
(pH 2.8) and dialysed into NaCl ⁄ P
i
. The eluate was passed
over a second column of the equivalent nonphosphopeptide
(EIGVAYGTVYKAC), the flow-through was collected and
dialysed into NaCl ⁄ P
i
containing 50% (v ⁄ v) glycerol.
96-well plate format CDK4 Y17 kinase assays

Half of the wells of Immulon 2HB 96-well plates (Dynex
Technologies Limited, Worthing, UK) were coated
with 1 lg per well of the CDK4 nonphosphopeptide
(EIGVAYGTVYKAC) at 4 °C overnight. Protein samples
were added to paired peptide-coated and noncoated wells,
and kinase buffer (50 mm Hepes, pH 7.4, 10 mm MgCl
2
,
1mm EGTA, 1 mm dithiothreitol, 0.4 mm NaF, 0.4 mm
Na
3
VO
4
,1mm ATP) was added to a total volume of
100 lL per well. The plate was incubated at 37 °C for
45 min and the reaction was stopped by washing the plate
three times in 0.1% (v ⁄ v) Tween-20. The plate was blocked
by incubation with 5% (w ⁄ v) skimmed milk powder in
TNT [50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.1% (v ⁄ v)
Tween-20] for 1 h. The CDK4 pY17 antibody diluted
1 : 1000 in 5% (w ⁄ v) skimmed milk powder in TNT was
added and incubated overnight at 4 °C. The plate was
washed three times in 0.1% (v ⁄ v) Tween-20 before antibody
detection using the DELFIAÒ Europium labelled anti-
rabbit secondary sera and a Wallac Victor 2 plate reader
(PerkinElmer, Waltham, MA, USA) as described by the
manufacturer. The counts from the nonpeptide-coated wells
were subtracted from the corresponding peptide-coated
wells, and the raw counts were used as the unit of kinase
activity. For the HeLa lysate PP2 inhibitor assay (Fig. 4B),

the ATP concentration was reduced to 50 lm and the dim-
ethylsulfoxide concentration was kept constant in all the
wells. For this assay, the SEM was converted to percent of
control and was calculated as: (1 ⁄ y)Ö[rx
2
+(x ⁄ y)
2
ry
2
],
where y is the sample set to 100%, x is the sample calcu-
lated relative to y, ry is the SEM of y and rx is the SEM
of x.
Tube format CDK4 Y17 kinase assay
Protein samples were mixed with kinase buffer (as above)
containing 5 lg of purified cyclin D1 ⁄ CDK4 complex [36]
and were incubated at 37 °C for 30 min. The CDK4 Y17
phosphorylation was detected by western blotting with the
CDK4 pY17 antibody.
Y17 kinase purification procedure
All protein purification steps were carried out at 4 °C and
all chromatography steps were performed using an AKTA
FPLC (Amersham Biosciences, GE Healthcare, Amersham,
UK). All chromatography columns and media were pur-
chased from Amersham Biosciences unless otherwise stated.
After each step, the Y17 kinase activity of each fraction
was measured using the 96-well plate format assay and the
protein concentration was assayed using Bradford reagent
(Bio-Rad, Hemel Hempstead, UK). Fractions from the
Superdex 200 and Hydroxyapatite columns were analysed

using the ATTO-TAG CBQCA protein assay (Invitrogen).
2 · 10
10
frozen HeLa Nuclei (Cil Biotech) with a mass of
approximately 80 g were lysed for 30 min in 400 mL of
KCl protein extraction buffer [50 mm Hepes, pH 7.4,
250 mm KCl, 1 mm EDTA, 0.1% (v ⁄ v) NP-40, 1 mm dith-
iothreitol] containing protease inhibitors (CompleteÔ
EDTA-free Protease Inhibitor Cocktail tablets; Roche
Diagnostics Ltd, Burgess Hill, UK) and phosphatase inhibi-
tors (10 mm b-glycerophosphate, 1 mm NaF, 0.1 mm
Na
3
VO
4
). The lysate was clarified by centrifugation at
15 000 g for 30 min followed by centrifugation at 100 000 g
for 1 h. Saturated ammonium sulfate solution (200 mL)
was added (33% final concentration) and incubated for
30 min. The precipitated proteins were collected by centri-
fugation at 2885 g for 30 min.
The protein pellets were dissolved in 400 mL of buffer A
[25 mm Hepes, pH 7.4, 0.6 m ammonium sulfate, 10%
(v ⁄ v) glycerol, 2 mm benzamidine hydrochloride, 1 mm
EDTA, 1 mm dithiothreitol] and clarified by centrifugation
at 100 000 g for 1 h, prior to loading onto an XK 50 ⁄ 30
column packed with Butyl Sepharose 4 Fast Flow (GE
Phosphorylation of CDK4 by Src family kinases N. G. Martin et al.
3106 FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS
Healthcare), equilibrated in buffer A. Proteins were eluted

with a 2 L linear gradient of 0.6–0 m ammonium sulfate
and 40 mL fractions were collected. Fractions 99–105 were
pooled for further purification.
The pooled elutes were dialysed into buffer B [25 mm
bis-tris, pH 7.0, 50 mm KCl, 10% (v ⁄ v) glycerol, 2 mm
benzamidine hydrochloride, 1 mm EDTA, 1 mm dithiothre-
itol], and loaded onto an XK 16 ⁄20 column packed with Q
Sepharose Fast Flow (GE Healthcare), equilibrated in
buffer B. Proteins were eluted with a 240 mL linear gradi-
ent of 50–400 mm KCl and 8 mL fractions were collected.
Chaps was added to the pooled elute to a final concen-
tration of 10 mm and the sample was concentrated from
24 mL to 4 mL using two Vivaspin 15R centrifugal concen-
trators (VWR International Ltd, Lutterworth, UK). The
concentrated sample was applied to a HiLoad 26 ⁄ 60 Super-
dex 200 pg gel filtration column (GE Healthcare) equili-
brated with buffer C [10 mm phosphate buffer, pH 7.0,
100 mm KCl, 10% (v ⁄ v) glycerol, 1 mm dithiothreitol,
10 mm Chaps], and proteins were eluted in the same buffer
and collected in 4 mL fractions.
The active Superdex 200 fractions were loaded onto a
Tricorn 5 ⁄ 50 column packed with 20 lm particle size Type
I CHT ceramic hydroxyapatite (Bio-Rad) equilibrated with
buffer C. The proteins were eluted with a linear gradient of
10–500 mm phosphate buffer and 500 lL fractions were
collected. 2.5 lL of sodium deoxycholate (2%, w ⁄ v) was
added to 250 lL samples of selected fractions and the sam-
ples were incubated for 15 min. 62.5 lL trichloroacetic acid
(50%, w ⁄ v) was added, the samples were incubated for a
further 1 h, and the proteins collected by centrifugation at

13000 g for 10 min. The precipitates were washed with ice-
cold ethanol, and the proteins were separated by
SDS ⁄ PAGE on a NuPAGE 4–12% Bis-Tris gradient gel in
Mops running buffer (Invitrogen). The proteins were
stained with Coomassie brilliant blue G (Sigma-Aldrich)
and a sample from the 60 kDa band was excised. The
sample was analysed using Q-TRAP MS by the Protein
Analysis Laboratory at the Cancer Research UK London
Research Institute (London, UK).
Immunoprecipitation
Cells were lysed in RIPA buffer [50 mm Hepes, pH 7.4,
150 mm NaCl, 1 mm EDTA, 1% (v ⁄ v) NP-40, 0.5% (w ⁄ v)
sodium deoxycholate, 0.1% (w ⁄ v) SDS, 1 mm dithiothrei-
tol] containing protease inhibitors (CompleteÔ EDTA-free
Protease Inhibitor Cocktail) and phosphatase inhibitors
(10 mm b-glycerophosphate, 1 mm NaF, 0.1 mm Na
3
VO
4
).
Insoluble debris was removed from the lysate by centrifuga-
tion at 13 000 g for 10 min and the protein concentration
was measured using Bradford reagent (Bio-Rad). Typically
lysates containing 1 mg of protein were incubated with
20 lL (bed volume) of Anti-FLAG M2 affinity gel (Sigma-
Aldrich) for 3 h at 4 °C, the beads were washed four times
with RIPA buffer and the precipitated proteins were
resolved by SDS ⁄ PAGE and western blotting.
Immunodepletion
Samples of HeLa whole cell and nuclear lysates prepared in

KCl protein extraction buffer containing 500 lg of protein
were depleted with 20 lg of anti-C-YES (Upstate), 5 lgof
anti-WEE1 H-300 (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) or 5 lg of anti-MYT1 N-17 (Santa Cruz
Biotechnology) polyclonal sera and 15 lL (bed volume) of
protein G-sepharose. For mock depletions, the antibody
was replaced with KCl protein extraction buffer. After
incubation with the beads and antibodies at 4 °C for 3 h,
the depleted supernatants were assayed for Y17 kinase
activity using both the 96-well plate format and tube
format assays. The beads were washed four times with KCl
protein extraction buffer and twice with kinase buffer
without ATP. The beads were then assayed for Y17 kinase
activity using the tube format assay.
Western blotting
Protein lysates and immunoprecipitates were resolved on
standard 10% SDS ⁄ PAGE gels, and chromatography frac-
tions were resolved on NuPAGE 4–12% Bis-Tris gradient
gels in Mops running buffer (Invitrogen). The proteins were
transferred onto Immobilon-P membranes [Millipore (UK)
Ltd, Watford, UK], which were blocked in 5% skimmed
milk powder in TNT. Membranes were incubated with
primary antibodies overnight and secondary antibodies
(peroxidase-conjugated goat anti-rabbit ⁄ mouse antibody;
Bio-Rad) for 1 h. Blots were developed using ECL Western
Blotting Detection Reagents and Hyperfilm (Amersham
Biosciences, GE Healthcare). Primary antibodies were
CDK4 C-22, WEE1 B-11, MYT1 N-17 (Santa Sruz
Biotechnology), CDK1 Ab-4 (NeoMarkers, Thermo Fisher
Scientific, Runcorn, UK), CDK1 phospho-Y15 (Cell

Signaling Technology, New England Biolabs, Hitchin,
UK), aTubulin DM1A (Sigma-Aldrich) and C-YES (BD
Biosciences, Oxford, UK).
Acknowledgements
We thank Jacky Metcalfe for the peptide synthesis,
Clive Lebozer for production of the rabbit antiserum,
and the Protein Analysis Laboratory at the Cancer
Research UK London Research Institute for perform-
ing the MS. We also thank the members of the Garrett
laboratory for useful discussion of the manuscript.
This work was supported by The Institute of Cancer
Research, Cancer Research UK (CUK) grant numbers
C309 ⁄ 2187 and C309 ⁄ A8274 and by AICR grant
02-112.
N. G. Martin et al. Phosphorylation of CDK4 by Src family kinases
FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3107
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FEBS Journal 275 (2008) 3099–3109 ª 2008 The Institute of Cancer Research: Royal Cancer Hospital. Journal compilation ª 2008 FEBS 3109