IkappaB kinase beta phosphorylates Dok1 serines in response to TNF, IL-1, or gamma radiation.

Abstract

Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that IkappaB kinase beta (IKKbeta) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKbeta phosphorylation of Dok1 depended on Dok1 S(439), S(443), S(446), and S(450). Recombinant IKKbeta also phosphorylated Dok1 or Dok1 amino acids 430-481 in vitro. TNF-alpha, IL-1, gamma radiation, or IKKbeta overexpression phosphorylated Dok1 S(443), S(446), and S(450) in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S(439), S(443), S(446), and S(450) mutated to A was not phosphorylated by IKKbeta in vivo. Surprisingly, mutant Dok1 A(439), A(443), A(446), and A(450) differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1/2 phosphorylation or cell growth. Mutant Dok1 A(439), A(443), A(446), and A(450) also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E(439), E(443), E(446), and E(450) further enhanced cell motility. These data indicate that IKKbeta phosphorylates Dok1 S(439)S(443) and S(446)S(450) after TNF-alpha, IL-1, or gamma-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.

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I?B kinase ? phosphorylates Dok1 serines in
response to TNF, IL-1, or ? radiation
Sanghoon Lee*†, Charlotte Andrieu*‡§, Fre ´de ´ric Saltel§¶, Olivier Destaing§¶, Jessie Auclair*?, Ve ´ronique Pouchkine*,**,
Jocelyne Michelon*, Bruno Salaun*††, Ryuji Kobayashi‡‡, Pierre Jurdic¶, Elliott D. Kieff§§, and Bakary S. Sylla*¶¶
*International Agency for Research on Cancer, 69008 Lyon, France;¶Ecole Normale Supe ´rieure, 69007 Lyon, France;‡‡University of Texas M. D. Anderson
Cancer Center, Houston, TX 77030; and§§Channing Laboratory, Harvard Medical School, Boston, MA 02115
Contributed by Elliott D. Kieff, October 29, 2004
Dok1 is an abundant Ras-GTPase-activating protein-associated
tyrosine kinase substrate that negatively regulates cell growth and
promotes migration. We now find that I?B kinase ? (IKK?) asso-
ciated with and phosphorylated Dok1 in human epithelial cells and
B lymphocytes. IKK? phosphorylation of Dok1 depended on Dok1
S439, S443, S446, and S450. Recombinant IKK? also phosphorylated
Dok1orDok1aminoacids430–481invitro.TNF-?,IL-1,?radiation,
or IKK? overexpression phosphorylated Dok1 S443, S446, and S450in
vivo, as detected with Dok1 phospho-S site-specific antisera. More-
over, Dok1 with S439, S443, S446, and S450 mutated to A was not
phosphorylated by IKK? in vivo. Surprisingly, mutant Dok1 A439,
A443, A446, and A450differed from wild-type Dok1 in not inhibiting
platelet-derived growth factor-induced extracellular signal-regu-
latedkinase1?2phosphorylationorcellgrowth.MutantDok1A439,
A443, A446, and A450 also did not promote cell motility, whereas
wild-type Dok1 promoted cell motility, and Dok1 E439, E443, E446,
and E450 further enhanced cell motility. These data indicate that
IKK? phosphorylates Dok1 S439S443and S446S450after TNF-?, IL-1,
or?-radiationandimplicatethecriticalDok1serinesinDok1effects
after tyrosine kinase activation.
NF-?B ? serine phosphorylation ? cell migration
D
growth factor receptor and nonreceptor tyrosine k inases (1, 2).
Related proteins include Dok2 (also known as FRIP or Dok-R),
Dok3 (also known as Dok-L), Dok4, Dok5, and insulin receptor
substrates (3–8). DOK proteins have an N-terminal pleckstrin
homology domain, a phosphotyrosine-binding (PTB) domain,
and a C terminus rich in proline, serine, and tyrosine (1, 2). The
pleckstrin homology domain mediates association with mem-
brane phospholipids, whereas the PTB domain mediates ho-
modimerization and association with phosphotyrosine signaling
molecules (9, 10). When it is tyrosine-phosphorylated, Dok1
interacts with other signaling molecules containing Src homol-
ogy 2 domains, such as Ras-GTPase-activating protein, SHIP1,
Nck, Csk, and SH2D1A (11–15).
DOK proteins down-modulate tyrosine kinase signaling ef-
fects. Dok1 can inhibit mitogen-activated protein kinase activa-
tion, cell proliferation, cell transformation, and leukemogenesis
(9, 10, 16–18). Dok1 can also affect cell adhesion, spreading,
migration, and apoptosis (13, 19, 20), and modulate T or B cell
receptor signaling (18, 21–24). Pleckstrin homology domain-
mediated plasma membrane translocation and tyrosine phos-
phorylation are critical for these Dok1 effects (10, 25).
After TNF-?, IL-1, or Toll receptor signaling, ? radiation, or
tyrosine kinase signaling, I?B kinase ? (IKK?) phosphorylates
I?B? S32S36, resulting in I?B? degradation, NF-?B activation,
and increased transcription of genes important for costimulatory
and survival effects (for reviews see refs. 15 and 26–37). Dok1
couldhavearoleinthephysiologicintegrationoftyrosinekinase
with TNF, IL-1, and Toll receptor signaling (38–41). In this
report, we investigate whether Dok1 S439S443and S446S450, which
are in a context similar to I?B? S32S36, can be phosphorylated by
ok1 or p62 dok is an abundant Ras-GTPase-activating
protein-associated adaptor protein that is downstream of
IKK? and potentially modify Dok1 effects downstream of ty-
rosine kinases.
Materials and Methods
Plasmids. pcDNA3-Flag-Dok1, pcDNA3-Myc-Dok1, and
pcDNA3-Flag-IKK??9 have been described (15, 42). Human
(Hu)Dok1 variants bearing specific mutations or deletions were
obtainedbyPCRmutagenesisandclonedintoexpressionvectors
(15). Dok1-SSSS is wild-type Dok1. Dok1-AAAA has S439, S443,
S446, and S450replaced by A. Mutants were cloned into expres-
sionplasmidsandsequence-confirmed.pRK5-Flag-IKK?(WT),
pRK5-Myc-IKK? (WT), and kinase-dead (KD) mutants, pRK5-
Flag-IKK? (KD) and pRK5-Myc-IKK? (KD) were obtained
from D. Goeddel (Tularik, South San Francisco, CA). The
expression plasmids for ataxia-telangiectasia-mutated (ATM)
were obtained from Y. Shiloh (Tel Aviv University, Tel Aviv)
and M. Kastan (St. Jude Children’s Research Hospital, Mem-
phis, TN). Glycogen synthetase kinase 3? expression plasmids
were obtained from J. Woodgett through G. Johnson (Ontario
Cancer Institute, Toronto). GFP-Dok1-SSSS was created by
inserting the coding sequences of the full-length cDNA of Dok1
in-frame with GFP into pEGFP-C1 (Clontech).
Reagents and Antibodies. TNF-?, IL-1, platelet-derived growth
factor (PDGF), anti-Flag M2, M5, and M2 affinity gels were
obtained from Sigma and R & D Systems. His-tagged recom-
binant IKK2 (IKK?) was provided by H. Allen (Abbott Biore-
search Center, Worcester, MA). ?-Phosphatase and leukocyte
antigen-related-tyrosine-phosphatase, monoclonal antibodies
against phospho-extracellular signal-regulated kinase (Erk)1?2
and phospho-I?B? were from New England Biolabs. IKK?- and
IKK?-specific, monoclonal phosphotyrosine, goat Erk1?2,
mouse phospho-I?B?, and rabbit I?B? antibodies were obtained
from Santa Cruz Biotechnology. Polyclonal antibodies against
phosphoserine (pS) 15 (S15) of p53 was obtained from Cell
Signaling Technology (Beverly, MA). Polyclonal rabbit anti-
Dok1 was described (1) and obtained from J. Cambier (St. Jude
Children’s Research Hospital). Rabbit polyclonal anti-phospho-
Dok1-Ser 439, -Ser 443, -Ser 446, and -Ser 450 antibodies were
Abbreviations: IKK?, I?B kinase ?; IKK?, I?B kinase ?; Erk, extracellular signal-regulated
kinase; pS, phosphoserine; HuDok, human Dok; MuDok, murine Dok; KD, kinase-dead; AT,
ataxia-telangiectasia; ATM, AT-mutated; PDGF, platelet-derived growth factor; HEK, hu-
man embryonic kidney; F-IKK?, Flag-tagged IKK?.
†Present address: Beth Israel Hospital, Harvard Medical School, Boston, MA 02115.
‡Presentaddress:InstitutNationaldelaSante ´ etdelaRechercheMe ´dicaleUnit450,Institut
Biome ´dical des Cordeliers, 75006 Paris, France.
§C.A., F.S., and O.D. contributed equally to this work.
?Present address: Centre d’Oncologie Ge ´ne ´tique, Centre Le ´on Be ´rard, 69008 Lyon, France.
**Present address: Complexe Universitaire des Ce ´seaux, 63174 Aubie `re, France.
††Present address: Schering–Plough Laboratory for Immunological Research, 69571 Dard-
illy, France.
¶¶To whom correspondence should be addressed at: International Agency for Research on
Cancer, 150 Cours Albert Thomas, 69008 Lyon, France. E-mail: sylla@iarc.fr.
© 2004 by The National Academy of Sciences of the USA
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raised against keyhole limpet hemocyanin-coupled peptides:
Dok1-pS439-SSS, H2N-CTG-pS-GIKSHNSALYSQVQK-
COOH; Dok1-S-pS443-SS, H2N-CTGSGIK-pS-HNSALYS-
QVQK-COOH; Dok1-SS-pS446-S, H2N-CTGSGIKSHN-pS-
ALYSQVQK-COOH; and Dok1-SSS-pS450, H2N-CTGSGIK-
SHNSALY-pS-QVQK-COOH by Covalab (Lyon, France).
Cell Culture and Transfections. Human embryonic kidney (HEK)
293, HEK 293 stably transfected with empty vector pcDNA3,
pcDNA3-Flag-Dok1-SSSS, pcDNA3-Flag-Dok1-AAAA,
pEGFP-C1, or pEGFP-Dok1-SSSS, and 293T cells were main-
tained in DMEM containing 10% FBS (D10). Clones expressing
Dok1 or containing empty vector, and polyclonal 293?GFP- or
293?GFP-Dok1-expressing cells were isolated after selection in
D10 containing 800 ?g?ml G418. Epstein–Barr virus immortal-
ization lymphoblastoid cell lines from a healthy individual
(lymphoblastoid cell line 2145) or from ataxia-telangiectasia
(AT) patients expressing a truncated ATM protein (AT-11 and
AT-14) were obtained from G. Lenoir (International Agency for
Research on Cancer) (43). These cell lines, as well as BJAB (a
Burkitt’s lymphoma cell line) and BJAB, stably transfected with
DNA3 or pcDNA3-Flag-IKK??9, were maintained in RPMI
medium 1640 supplemented with 10% FBS. Cells were trans-
fected by electroporation or by Superfect (Qiagen, Valencia,
CA) (15). To determine growth curves, cells were seeded in
six-well plates at a density of 1 ? 104and cultured in D10. The
medium was changed every 48 h and the number of cells was
counted every 24 h. For cytokine stimulation and ionizing
radiation, cells were treated with TNF-? (20 ng?ml), IL-1 (20
ng?ml), or PDGF (20 ng?ml), or were exposed to ? radiation (20
Gy) by using a137Ce IBL-437c irradiator (CIS Biointernational,
Gif-sur-Yvette, France), and harvested at the indicated time
points.Equalamountsofproteinfromthetotalcellextractswere
analyzed by Western blot.
Cell Spreading and Wound Healing. For cell spreading assays, 293
cells stably transfected were seeded at a density of 105cells per
ml in 60-mm-diameter culture dishes coated with collagen type
I (10 ?g?ml, Sigma). After incubation at 37°C, the cells were
examined under a light microscope equipped with phase-
contrast optics (model IX 70, Olympus) and photographed. For
the wounding test, cells were seeded on 35-mm-diameter dishes
precoated with collagen type I and grown to confluence in the
presence of serum. The medium was changed every 24 h. The
wound was made by scraping across the center of the dish with
0.1- to 2-?l tips. The medium was changed after scraping to
eliminate nonadherent cells. The cells were then incubated at
37°C and examined at different times by using a Leica (Ban-
nockburn, IL) DM IRB microscope (objective N plan ?10). The
area of the wound was measured in pixels by using METAMORPH
software (Universal Imaging, West Chester, PA).
Immunoprecipitation, GST Pull-Down Assays, and Western Blotting.
Immunoprecipitation, GST pull-down assays, and Western blot
analyses were described (15).
Assay of Erk Activation and Kinase Assays. Cells were deprived of
serum for 24 h and then incubated with PDGF (20 ng?ml,
Calbiochem) for indicated times. Antibodies specific for acti-
vated Erks and total Erks were used for immunoblotting. The in
vitro kinase assays were described (44).
Confocal Microscopy and Immunofluorescence. Cells were seeded in
35-mm glass-bottom Petri dishes coated with collagen I, and
immunofluorescence was as described (45). F-actin distribution
was observed after incubation of cells with rhodamine B iso-
thiocyanate-conjugated phalloidin (Molecular Probes) at 0.4
units?ml for 20 min at room temperature. Samples were imaged
with a Zeiss LSM 510 microscope by using a ?63 (numerical
aperture of 1.4) Plan Neo Fluor objective.
Results
IKK? Associates with and Phosphorylates Dok1. To test whether
Dok1 associates with IKK?, Myc-tagged Dok1 and Flag-tagged
IKK? (F-IKK?) were expressed in 293T cells (Fig. 1A). Approxi-
mately 0.5–1% of Dok1 in the cell lysates coprecipitated with
?10–20% of F-IKK?. The coimmunoprecipitation was specific
because Dok1 was not precipitated from cells in which F-IKK? was
notexpressed(Fig.1A).Furthermore,?0.1%ofendogenousDok1
specifically associated with ?20% of IKK? in F-IKK? immuno-
precipitates from lysates of BJAB cells constitutively expressing
F-IKK? (Fig. 1B) or with ?4% of endogenous IKK? in nontrans-
fected BJAB cells (Fig. 1C). Thus, corrected for the efficiency of
IKK? immunoprecipitation, 0.5–5% of Dok1 is stably associated
with IKK? in 293T epithelial cells or BJAB B lymphoblasts. By
using polyclonal Dok1 antibody to immunoprecipitate Dok1 from
BJAB B lymphoblasts, ?0.5% of IKK? appeared to be stably
Dok1-associated, after correction for the ?20% efficiency of Dok1
precipitation (Fig. 1C); polyclonal antibody could interfere with or
inhibit intermolecular associations. Moreover, pervanadate inhibi-
tion of phosphatases substantially increased Dok1 tyrosine phos-
phorylation and resulted in almost 2-fold more Dok1 or phospho-
tyrosine-Dok1associationwithoverexpressedF-IKK?in293Tcells
(Fig. 1A). Interestingly, IKK? overexpression decreased Dok1
tyrosine phosphorylation (Fig. 1A), which is consistent with the
possibility that IKK? may down-modulate Dok1 effects down-
stream of tyrosine kinases.
Fig. 1.
cells were transfected with expression plasmids for Myc-Dok1 (Dok1) and
Flag-IKK??9 (F-IKK?). After 48 h, half of the cultures were treated with
pervanadate (PV). Immunoprecipitates were analyzed by immunoblotting. L,
1% lysate; F, 30% of immunoprecipitate; IgG, Ig heavy chain. (B) Protein
extracts from BJAB cells stably expressing Flag-IKK??9 were immunoprecipi-
tated and analyzed as in A. (C) Protein extracts from BJAB cells were immu-
noprecipitated with mouse anti-IKK? or rabbit anti-Dok1. Immunoblotting
wasperformedbyusingmouseanti-IKK?,rabbitanti-IKK?,orrabbitanti-Dok.
A faint band above IKK? may represent IKK? due to crossreactivity. (D) HEK
293Tcells were cotransfected with vectors expressing Myc-Dok1 and Flag-
IKK?. After 24 h, cells were fixed. IKK? was visualized with mouse anti-IKK?
and Dok1 with rabbit anti-Dok1. F-actin was visualized with rhodamine
isothiocyanate-conjugated phalloidin. Colors are confocal interface false col-
ors with F-actin-labeled phalloidin excited at 546 nm and IKK?-labeled cyanin
5 excited at 633 nm. Arrowheads in the merge image indicate Dok1 and IKK?
colocalization. Data are representative of at least three independent
experiments.
IKK? associates with Dok1, inducing a mobility shift. (A) HEK 293T
Lee et al.
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In transfected HEK 293T cells, overexpressed Dok1 and IKK?
were distributed in a reticular pattern throughout the cytoplasm,
with enhancement near the plasma membrane and in cytoplas-
mic extensions, near sites of F-actin accumulation (Fig. 1D and
data not shown).
IKK?overexpressionwithDok1resultedinaslowermigrating
Dok1 fraction (Fig. 2A). The mobility shift occurred with native,
Flag-, or Myc- tagged Dok1 (data not shown). The fraction of
Dok1 shift varied directly with the amount of IKK? expression
vector and the amount of IKK? expressed (Fig. 2A), implicating
IKK? in Dok1 modification. I?B kinase ? (IKK?) overexpres-
sion also induced similar Dok1 modification, whereas KD
IKK?(KD)orIKK?(KD)didnotinduceDok1modification(Fig.
2B). Moreover, F-IKK?(KD) or M-IKK?(KD) had dominant
negative effects on wild-type F-IKK?-induced Dok1 modifica-
tion (data not shown). Furthermore, Yersinia YopJ, an inhibitor
of IKK? and other mitogen-activated protein kinases (46),
prevented IKK?-induced Dok1 modification (Fig. 2C). The
Dok1 mobility shift disappeared with ?-Ser?Thr phosphatase
treatment, but not with leukocyte antigen-related tyrosine phos-
phatase treatment (Fig. 2D). Thus, increased IKK? or IKK?
activity causes Dok1 Ser?Thr phosphorylation.
To assess whether Dok1 is a direct IKK? substrate, IKK? was
expressed and purified from HEK 293 cells or from Sf9 insect
cells,andincubatedinaninvitrokinasereactionwithEscherichia
coli-expressed putative substrates, GST-Dok1, GST-Dok1
amino acids 430–481, GST-I?B? positive control, and GST-
I?BaA32A36negative control proteins. IKK? from 293 or Sf9
cells specifically phosphorylated GST-I?B? and, to a lesser
extent, GST-Dok1, GST-Dok1 430–481, and IKK?, whereas
IKK? failed to phosphorylate GST- I?BaA32A36(Fig. 8, which
is published as supporting information on the PNAS web site).
Overexpression of mitogen-activated protein kinase?Erk kinase
kinase 1, an upstream activator of IKK (47) also induced Dok1
mobility shift in 293T cells (data not shown). These data indicate
that IKK? can directly phosphorylate Dok1 or Dok1 amino acids
430 and 481 and that Dok1 amino acids 430–481 are a sufficient
substrate for IKK? phosphorylation.
IKK? Phosphorylation Sites in Dok1. HuDok1 S439S443and S446S450
and the homologous sites in murine (Mu)Dok1 and HuDok2 are
separated by three amino acids, as are the critical serines for
IKK? phosphorylation sites in I?B?, ?, ?, and Cactus (Fig. 3A).
HuDok1lacksaDbeforeS439,S443,S446,orS450,andthisfactmay
be the basis for less efficient phosphorylation, relative to I?B?,
?, ?, and Cactus (Fig. 8). However, Hu and MuDok1 have a Y
before S450, and phosphorylation of that tyrosine may mimic D
or E in I?B? and I?B?, respectively, and account for the
increased association of tyrosine-phosphorylated Dok1 with
IKK? in Fig. 1A. The increased association could also be due to
Dok1 dimerization after tyrosine phosphorylation.
The role of Dok1 S439S443S446S450in IKK? phosphorylation
was further evaluated by determining whether IKK? overex-
pression could induce the phosphorylation of Dok1
A439A443A446A450 (Fig. 3B). IKK? overexpression resulted in
substantial Dok1 phosphorylation and slower mobility, whereas
Dok1 A439A443A446A450was not detectably phosphorylated, and
Dok1 with A substituted for any three of the four Ss was not
extensively phosphorylated (Fig. 3B). Thus, Dok1 S439S443S446,
and S450are critical for IKK? phosphorylation. However, Dok1
with As substituted for the first or last two Ss was intermediate
in the intensity of highly phosphorylated Dok1, indicating that
S439S443and S446S450can independently enable extensive phos-
phorylation with reduced efficiency (Fig. 3B). These data are
most consistent with the possibility that IKK?-induced Dok1 S/T
phosphorylation extends sequentially beyond S439–S450.
Dok1 pS443- or pS450-specific antibodies were raised in rabbits
purified on phosphopeptide affinity columns and readily de-
tected phosphorylated Dok1 in extracts from 293T cells after
overexpression of IKK? and Dok1 (Fig. 4). As expected, phos-
phospecific antibody reactivity was not detected with overex-
pression of IKK? and Dok1 A439A443A446A450 (Fig. 4). Dok1
antibody detected Dok1 and Dok1 A439A443A446A450 equally
(Fig. 4). Antibodies to pS443and pS450reacted at low levels with
wild-type Dok1, but not with Dok1 A439A443A446A450 in the
absence of cotransfected IKK? (Fig. 4). Some of this low-level
reactivity is likely due to phosphorylation by endogenous IKK,
given the high antibody specificity for Dok1 over Dok1
A439A443A446A450. However, pS450antibody did have low non-
phosphospecific reactivity with Dok1 A450, whereas antibody to
pS443appearedtolackreactivitywithDok1A443(Fig.4).Overall,
these data indicate that antibody to pS443 and pS450 have
substantial specificity and that IKK? overexpression causes
Dok1 phosphorylation at these sites. Similar specificity was
Fig.2.
and increasing amounts of F-IKK? expression vector DNAs. Cell lysates were
analyzedbyWesternblotting.*,IKK?-modifiedDok1.(B)HEK293Tcellswere
transfectedwithF-Dok1togetherwithF-IKK?,F-IKK?KD,F-IKK?,orF-IKK?KD
expression vector DNAs. (C) F-Dok1, F-IKK?, and F-YopJ expression vector
DNAs were transfected into 293T cells and cell lysates were analyzed by
Westernblot.(D)HEK293TCellsweretransfectedwithF-Dok1withorwithout
F-IKK? expression vector DNA. Aliquots of whole-cell lysates were treated
with Ser?Thr phosphatase (?-PPase) or tyrosine phosphatase (leukocyte
antigen-related-Tyr-PPase), and proteins were analyzed by Western blot.
Similar results were obtained in three independent experiments.
IKK?isaDok1kinase.(A)HEK293TcellsweretransfectedwithF-Dok1
Fig. 3.
serines that are IKK?-phosphorylated are compared with HuDok1 S439, S443,
S446, and S450, with MuDok1, and with HuDok2. (B) HEK 293T cells were
transfectedwithwild-typeDok1(F-Dok1-SSSS)orvariousalaninesubstitution
mutants, with or without F-IKK?. Total cell lysates were analyzed by Western
blot using anti-Dok1 or anti-IKK? antibodies. Data are representative of two
or more independent experiments.
Dok1 is an IKK? substrate. (A) I?B?, I?B?, I?B?, and Drosophila Cactus
17418 ?
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obtained with antibody to peptide pS446, but not with antibody
to peptide pS439(data not shown).
TNF-?, IL-1, and ? Irradiation Induce IKK?-Mediated Dok1 Phosphor-
ylation. To determine whether IKK?-mediated Dok1 phosphor-
ylation occurs after physiologic IKK? activation, Flag-Dok1 or
Flag-Dok1 AAAA stably expressing 293 cells were stimulated by
TNF-? or IL-1, potent IKK? activators. Whereas total Dok1
levels were similar in most clones and did not change after
TNF-? or IL-1 treatment, TNF-? or IL-1 consistently induced
pS443and pS450reactivity within 10 min. Phosphorylation was
maximal at 10–20 min and persisted at nearly maximal levels for
at least 30 min (Fig. 5A and data not shown for pS450). As
expected, TNF-? or IL-1 did not cause Dok1-AAAA phosphor-
ylation, as was evident by the absence of change in Dok1 pS443
or pS450antibody reactivity in Western blots of 293 cell extracts
(data not shown). TNF-? also induced endogenous Dok1 S443
and S450 phosphorylation by 10 min in HeLa cells (Fig. 5B).
Moreover, overexpression of dominant-negative IKK?(KD) in-
hibited TNF-? induced Dok1 phosphorylation in 293 cells
expressing F-Dok1 (data not shown).
Ionizing radiation also activated IKK? (48, 49) and induced
Dok1 pS450reactivity, which reached its maximum at 1–2 h and
persisted for at least 6 h (Fig. 5D). Radiation-induced phosphor-
ylated Dok1 had an electrophoretic mobility similar to that
observed with IKK? overexpression (data not shown). Phos-
phorylation of S443was also induced, but to lesser extent (data
not shown). Ionizing radiation-induced IKK?-mediated NF-?B
activationdependsonthekinaseATMthatismutatedinAT(48,
49). As expected, ATM was required for Dok1 phosphorylation,
and Dok1 S450was not phosphorylated in lymphoblastoid cell
lines from two AT patients (Fig. 5D; AT-11 and AT-14) har-
boring a nonfunctional truncated ATM protein (43). Thus,
ionizing radiation-induced Dok1 phosphorylation is ATM-
dependent. Phosphorylation of p53 at S15was observed in AT
mutant cell lines at 20 Gy because other ATM family members
such as ATR can phosphorylate p53 (50). These data indicate
that ATM is the principal kinase for the initiation of Dok1
phosphorylation after ionizing radiation exposure. We cannot
exclude the possibility that ATM also directly phosphorylates
Dok1 because Dok1 S310and S450are potential SQ?TQ ATM
phosphorylation motif sites (1, 50). In fact, ATM overexpression
induced Dok1 pS450reactivity, but the extent of phosphorylation
was much less than that induced by IKK? or ionizing radiation
(data not shown). Taken together, these data indicate that TNF-,
IL-1-, or ? radiation-induced IKK? activation results in Dok1
S443,S450phosphorylation.
Dok1 S439, S443, S446, and S450Are Required for Dok1 Effects on Cell
Proliferation,onPDGF-InducedErk1?2Activation,andonCellMotility.
Wild-type Dok1 overexpression down-modulates cell prolifera-
tion and mitogen-activated protein kinase activation (16). The
role of the Dok1 IKK? phosphorylation sites in Dok1 effects on
cell proliferation and Erk1?2 kinase activity in 293 cells was
evaluated by comparing the effects of stable Dok1 or Dok1-
AAAA overexpression on cell proliferation and Erk1?2 kinase
activity. As expected (16), cells overexpressing Dok1 grew more
slowly than control cells (Fig. 6A). In contrast, cells expressing
similar amounts of Dok1-AAAA grew at a rate that was
indistinguishable from control cells (Fig. 6A). PDGF also in-
duced less Erk1?2 phosphorylation in cells that overexpress
wild-type Dok1, whereas PDGF-induced Erk1?2 phosphoryla-
tion was normal or elevated in cells that overexpress Dok1-
AAAA (Fig. 6B). Thus, the Dok1 serines that are phosphory-
lated by IKK? are required for Dok1 overexpression-mediated
inhibition of Erk1?2 phosphorylation and cell proliferation,
which is consistent with a role for IKK?-mediated phosphory-
lation in Dok1 effects downstream of tyrosine kinases.
As described (13, 18, 19), Dok1 overexpression increased 293
cell spreading and migration on coverslips coated with collagen
I (Fig. 7A). Although polyclonal cells converted to GFP expres-
sion were indistinguishable from polyclonal cells expressing
GFP-Dok1, in their growth on plastic (except for GFP-Dok1
localization to the cytoplasm and GFP distribution throughout
the cell), cells expressing GFP-Dok1 became flatter and more
spread out than GFP-expressing cells within hours of plating on
a collagen-I substrate in complete medium with serum (Fig. 7A
Left). Furthermore, GFP-Dok1-expressing cells migrated more
rapidly into a wound in the cell layer than did GFP control cells
(Fig. 7B Right). In contrast, Flag-Dok1-AAAA-converted cells
Fig. 4.
transfected with an expression vector for F-Dok1-SSSS, AAAA, ASSS, SASS,
SSAS,orSSSAwithorwithoutF-IKK?.Dok1phosphorylationwasevaluatedby
using anti-pS443 or anti-pS450 affinity-purified antibody. Dok1 and IKK?
levels were monitored by Western blot. These data are representative of at
least three independent experiments.
Dok1 pS443- and pS450-specific antibodies. HEK 293T cells were
Fig.5.
cells stably transfected with F-Dok1-SSSS were treated with TNF-? (20 ng?ml)
or IL-1 (20 ng?ml), and protein extracts were analyzed by immunoblot using
Dok1 and Dok1 pS443. (B) HeLa cells were treated with TNF-? (20 ng?ml) for
10 min. Protein extracts were immunoprecipitated with rabbit antibody to
Dok1 and analyzed by immunoblot with Dok1 and Dok1 pS443 or pS450
antibody. (C and D) BJAB or Epstein–Barr virus-transformed lymphoblasts
fromanormalcontrol(lymphoblastoidcellline2145)orfromtwopeoplewith
AT (AT11 and AT14) were exposed to ? radiation (20 Gy) and equal protein
extracts were immunoblotted with Dok1 and Dok1 pS450, or p53 pS15. These
data are representative of two or more independent experiments.
TNF-?,IL-1,or?radiationinducesDok1pS443andpS450.(A)HEK293
Lee et al.
PNAS ?
December 14, 2004 ?
vol. 101 ?
no. 50 ?
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CELL BIOLOGY

Page 5

were significantly slower in migration than Flag-Dok1 cells (Fig.
7B Left). Moreover, cells expressing Dok1-SSEE or Dok1-EEEE
migrated significantly faster than did wild-type Dok1-expressing
cells (Fig. 7B Right). These data implicate Dok1 S439, S443, S446,
and S450in Dok1 effects on cell spreading and migration, which
is consistent with previously observed TNF-?- or IL-1-induced
increase in cell spreading; such effects have also been attributed
to other mitogen-activated protein kinase pathways (51–53).
Importantly, Dok1 SSSS or AAAA overexpression did not
affect IKK?-mediated I?B? phosphorylation or subsequent
degradation after TNF-? treatment (data not shown), indicating
that Dok1 does not compete with I?B? for IKK?, even when
Dok1 is overexpressed.
Discussion
These experiments indicate that physiologic IKK? activation
results in Dok1 S443, S446, and S450phosphorylation. IKK? (and
?) associated with and phosphorylated Dok1 on S439S443 and
S446S450. Extensive IKK?-mediated Dok1 phosphorylation, in
vivo,dependedonS439,S443,S446,andS450,andDok1aminoacids
430–481 were phosphorylated by recombinant IKK?, in vitro.
Furthermore, TNF-?, IL-1, or ionizing radiation induced IKK?
activation and Dok1 S443, S446, and S450phosphorylation, in vivo,
as assayed with Dok1 pS site-specific antisera. Moreover, Dok1
phosphorylation after ionizing radiation was ATM-dependent,
which was consistent with the expected ATM role in activating
IKK?. Dok1 S443, S446, and S450phosphorylations were down-
stream of TNF-?, IL-1, or ionizing radiation-induced IKK?
activation in human epithelial and B cells.
Dok1 was a less efficient than I?B? as an in vitro substrate for
activated IKK?. Furthermore, Dok1 overexpression did not
affect IKK?-mediated NF-?B activation. Dok1 therefore ap-
pears to be an alternative IKK? substrate that does not inhibit
IKK? phosphorylation of I?B?.
The data presented here support the hypothesis that Dok1
S439,S443,S446, and S450, which are phosphorylated by IKK?, are
critical for previously characterized Dok1 effects downstream of
tyrosine kinase signaling. Activated IKK? phosphorylation of
these Dok1 serines may augment Dok1 effects. Indeed, mutation
of Dok1 S439, S443, S446, and S450to A resulted in Dok1 being
unable to inhibit PDGF-mediated Erk1?2 activation or cell
proliferation.
Most relevant to a role for Dok1 serine phosphorylation were
motility studies in which wild-type Dok1 overexpression in-
creased cell motility, Dok1 with S439, S443, S446, and S450mutated
to A439, A443, A446, and A450was unable to increase cell motility,
and Dok1 with S439, S443, S446, and S450mutated to E439, E443,
E446, and E450was slightly superior to wild-type Dok1 in increas-
ing cell motility. Moreover, as shown in Fig. 1D, Dok1 and IKK?
had a diffuse reticular distribution, with accentuation at sites of
F-actin, which is consistent with a role for IKK? in filopodia
formation, possibly related to cell motility. Altogether, these
biochemical and reverse genetic data are consistent with the
hypothesis that IKK? phosphorylation of Dok1 is important for
Dok1 effects on cell motility.
Other evidence supports a role for Dok1 in cell motility.
Mu-Dok1 Y361, which is equivalent to Hu-Dok1 Y362, is also a
specific c-Abl substrate in fibroblasts and mediates Nck recruit-
ment followed by Pak1 or mitogen-activated protein kinase Erk
kinase kinase 1 activation (13, 54–57). Mouse fibroblasts lacking
c-Abl, Dok1, or Nck have fewer filopodia than cells expressing
the disrupted gene (57).
Dok1 is now among a growing list of ‘‘nonclassical’’ IKK?
substrates. Dok1 S439S443and S446S450are similar in amino acid
sequence to I?B? S32S36. Furthermore, Dok1 Y449is an impor-
tant Csk tyrosine kinase site and Y449phosphorylation next to
Fig. 7.
Dok1-AAAA inhibits cell migration. (A) Polyclonal selected 293?GFP-or 293?
GFP-Dok1-expressing cells were plated in serum on coverslips coated with
collagenI.Cellswereobservedbyfluorescence(FL)andphasecontrast(Ph)for
wound healing (WH) 2 h later. Healing (WH) in the cell layer was photo-
graphed for the indicated cell lines at 0 (WH 0) and 2 h (WH 2). Cell migration
was evaluated based on cell filling of the wound area by using METAMORPH
software. (B) Wound healing test was performed as in A for cell lines express-
ing Flag-tagged Dok1 wild-type (WT), Dok1-AAAA mutant (AAAA), Dok1-
EESS, Dok1-SSEE, or Dok1-EEEE. The percentage filling at 2 h for each cell line
isplotted.Dataaremeans?SEofthreeindependentexperimentscarriedout
in triplicate.
Dok1, Dok1-SSEE, and Dok1-EEEE increase cell migration, whereas
Fig.6.
Dok1-AAAA does not affect cell proliferation and slightly increases Erk1?2
phosphorylation.(A)HEK293cellsstablytransfectedwithDok1,Dok1-AAAA,
or empty pcDNA3 expression vectors were monitored for growth. Data are
mean ? SE of three independent experiments carried out in triplicate. Similar
results were also obtained from two other independent sets of expressing
clones. (B) Equal protein extracts from PDGF-treated pcDNA3-, Dok1-, or
Dok1-AAAA-transfected cells lines were analyzed by immunoblotting for
pERK1?2, Erk1?2, and Dok1. Data are representative of at least three inde-
pendent experiments.
Dok1inhibitscellproliferationandErk1?2phosphorylation,whereas
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www.pnas.org?cgi?doi?10.1073?pnas.0408061101Lee et al.