IB kinase  phosphorylates Dok1 serines in response to TNF, IL-1, or ␥ radiation
Sanghoon Lee*†, Charlotte Andrieu*‡§, Fre´ de´ ric Saltel§¶, Olivier Destaing§¶, Jessie Auclair*ʈ,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 IKK and potentially modify Dok1 effects downstream of ty- tyrosine kinase substrate that negatively regulates cell growth and rosine kinases. promotes migration. We now find that IB kinase  (IKK) asso- ciated with and phosphorylated Dok1 in human epithelial cells and Materials and Methods B lymphocytes. IKK phosphorylation of Dok1 depended on Dok1 Plasmids. pcDNA3-Flag-Dok1, pcDNA3-Myc-Dok1, and S439,S443,S446, and S450. Recombinant IKK also phosphorylated pcDNA3-Flag-IKK⌬9 have been described (15, 42). Human Dok1 or Dok1 amino acids 430–481 in vitro. TNF-␣, IL-1, ␥ radiation, (Hu)Dok1 variants bearing specific mutations or deletions were or IKK overexpression phosphorylated Dok1 S443,S446, and S450 in obtained by PCR mutagenesis and cloned into expression vectors vivo, as detected with Dok1 phospho-S site-specific antisera. More- (15). Dok1-SSSS is wild-type Dok1. Dok1-AAAA has S439,S443, over, Dok1 with S439,S443,S446, and S450 mutated to A was not S446, and S450 replaced by A. Mutants were cloned into expres- phosphorylated by IKK in vivo. Surprisingly, mutant Dok1 A439, sion plasmids and sequence-confirmed. pRK5-Flag-IKK (WT), A443,A446, and A450 differed from wild-type Dok1 in not inhibiting pRK5-Myc-IKK␣ (WT), and kinase-dead (KD) mutants, pRK5- platelet-derived growth factor-induced extracellular signal-regu- Flag-IKK (KD) and pRK5-Myc-IKK␣ (KD) were obtained lated kinase 1͞2 phosphorylation or cell growth. Mutant Dok1 A439, from D. Goeddel (Tularik, South San Francisco, CA). The A443,A446, and A450 also did not promote cell motility, whereas expression plasmids for ataxia-telangiectasia-mutated (ATM) wild-type Dok1 promoted cell motility, and Dok1 E439,E443,E446, were obtained from Y. Shiloh (Tel Aviv University, Tel Aviv) and E450 further enhanced cell motility. These data indicate that and M. Kastan (St. Jude Children’s Research Hospital, Mem- IKK phosphorylates Dok1 S439S443 and S446S450 after TNF-␣, IL-1, phis, TN). Glycogen synthetase kinase 3 expression plasmids or ␥-radiation and implicate the critical Dok1 serines in Dok1 effects were obtained from J. Woodgett through G. Johnson (Ontario after tyrosine kinase activation. Cancer Institute, Toronto). GFP-Dok1-SSSS was created by inserting the coding sequences of the full-length cDNA of Dok1 NF-B ͉ serine phosphorylation ͉ cell migration in-frame with GFP into pEGFP-C1 (Clontech). ␣ ok1 or p62 dok is an abundant Ras-GTPase-activating Reagents and Antibodies. TNF- , IL-1, platelet-derived growth Dprotein-associated adaptor protein that is downstream of factor (PDGF), anti-Flag M2, M5, and M2 affinity gels were growth factor receptor and nonreceptor tyrosine k inases (1, 2). obtained from Sigma andR&DSystems. His-tagged recom-  Related proteins include Dok2 (also known as FRIP or Dok-R), binant IKK2 (IKK ) was provided by H. Allen (Abbott Biore- Dok3 (also known as Dok-L), Dok4, Dok5, and insulin receptor search Center, Worcester, MA). -Phosphatase and leukocyte substrates (3–8). DOK proteins have an N-terminal pleckstrin antigen-related-tyrosine-phosphatase, monoclonal antibodies homology domain, a phosphotyrosine-binding (PTB) domain, against phospho-extracellular signal-regulated kinase (Erk)1͞2 and a C terminus rich in proline, serine, and tyrosine (1, 2). The and phospho-IB␣ were from New England Biolabs. IKK␣- and pleckstrin homology domain mediates association with mem- IKK-specific, monoclonal phosphotyrosine, goat Erk1͞2, brane phospholipids, whereas the PTB domain mediates ho- mouse phospho-IB␣, and rabbit IB␣ antibodies were obtained modimerization and association with phosphotyrosine signaling from Santa Cruz Biotechnology. Polyclonal antibodies against molecules (9, 10). When it is tyrosine-phosphorylated, Dok1 phosphoserine (pS) 15 (S15) of p53 was obtained from Cell interacts with other signaling molecules containing Src homol- Signaling Technology (Beverly, MA). Polyclonal rabbit anti- ogy 2 domains, such as Ras-GTPase-activating protein, SHIP1, Dok1 was described (1) and obtained from J. Cambier (St. Jude Nck, Csk, and SH2D1A (11–15). Children’s Research Hospital). Rabbit polyclonal anti-phospho- DOK proteins down-modulate tyrosine kinase signaling ef- Dok1-Ser 439, -Ser 443, -Ser 446, and -Ser 450 antibodies were fects. Dok1 can inhibit mitogen-activated protein kinase activa- tion, cell proliferation, cell transformation, and leukemogenesis Abbreviations: IKK,IB kinase ; IKK␣,IB kinase ␣; Erk, extracellular signal-regulated (9, 10, 16–18). Dok1 can also affect cell adhesion, spreading, kinase; pS, phosphoserine; HuDok, human Dok; MuDok, murine Dok; KD, kinase-dead; AT, migration, and apoptosis (13, 19, 20), and modulate T or B cell ataxia-telangiectasia; ATM, AT-mutated; PDGF, platelet-derived growth factor; HEK, hu- receptor signaling (18, 21–24). Pleckstrin homology domain- man embryonic kidney; F-IKK, Flag-tagged IKK. mediated plasma membrane translocation and tyrosine phos- †Present address: Beth Israel Hospital, Harvard Medical School, Boston, MA 02115. phorylation are critical for these Dok1 effects (10, 25). ‡Present address: Institut National de la Sante´et de la Recherche Me´dicale Unit 450, Institut After TNF-␣, IL-1, or Toll receptor signaling, ␥ radiation, or Biome´dical des Cordeliers, 75006 Paris, France. tyrosine kinase signaling, IB kinase  (IKK) phosphorylates §C.A., F.S., and O.D. contributed equally to this work. ʈ IB␣ S32S36, resulting in IB␣ degradation, NF-B activation, Present address: Centre d’Oncologie Ge´ne´ tique, Centre Le´onBe´ rard, 69008 Lyon, France. and increased transcription of genes important for costimulatory **Present address: Complexe Universitaire des Ce´seaux, 63174 Aubie`re, France. and survival effects (for reviews see refs. 15 and 26–37). Dok1 ††Present address: Schering–Plough Laboratory for Immunological Research, 69571 Dard- could have a role in the physiologic integration of tyrosine kinase illy, France. with TNF, IL-1, and Toll receptor signaling (38–41). In this ¶¶To whom correspondence should be addressed at: International Agency for Research on report, we investigate whether Dok1 S439S443 and S446S450, which Cancer, 150 Cours Albert Thomas, 69008 Lyon, France. E-mail: [email protected]. are in a context similar to IB␣ S32S36, can be phosphorylated by © 2004 by The National Academy of Sciences of the USA
17416–17421 ͉ PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408061101 Downloaded by guest on September 26, 2021 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 293͞GFP- or 293͞GFP-Dok1-expressing cells were isolated after selection in D10 containing 800 g͞ml 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  ⌬ Fig. 1. IKK associates with Dok1, inducing a mobility shift. (A) HEK 293T DNA3 or pcDNA3-Flag-IKK 9, were maintained in RPMI cells were transfected with expression plasmids for Myc-Dok1 (Dok1) and medium 1640 supplemented with 10% FBS. Cells were trans- Flag-IKK⌬9 (F-IKK). After 48 h, half of the cultures were treated with fected by electroporation or by Superfect (Qiagen, Valencia, pervanadate (PV). Immunoprecipitates were analyzed by immunoblotting. L, CA) (15). To determine growth curves, cells were seeded in 1% lysate; F, 30% of immunoprecipitate; IgG, Ig heavy chain. (B) Protein six-well plates at a density of 1 ϫ 104 and cultured in D10. The extracts from BJAB cells stably expressing Flag-IKK⌬9 were immunoprecipi- medium was changed every 48 h and the number of cells was tated and analyzed as in A.(C) Protein extracts from BJAB cells were immu- counted every 24 h. For cytokine stimulation and ionizing noprecipitated with mouse anti-IKK or rabbit anti-Dok1. Immunoblotting ␣ ͞ was performed by using mouse anti-IKK, rabbit anti-IKK␣, or rabbit anti-Dok. radiation, cells were treated with TNF- (20 ng ml), IL-1 (20 ␣  ng͞ml), or PDGF (20 ng͞ml), or were exposed to ␥ radiation (20 A faint band above IKK may represent IKK due to crossreactivity. (D) HEK 137 293Tcells were cotransfected with vectors expressing Myc-Dok1 and Flag- Gy) by using a Ce IBL-437c irradiator (CIS Biointernational, IKK. After 24 h, cells were fixed. IKK was visualized with mouse anti-IKK Gif-sur-Yvette, France), and harvested at the indicated time and Dok1 with rabbit anti-Dok1. F-actin was visualized with rhodamine points. Equal amounts of protein from the total cell extracts were isothiocyanate-conjugated phalloidin. Colors are confocal interface false col- analyzed by Western blot. 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 Cell Spreading and Wound Healing. For cell spreading assays, 293 colocalization. Data are representative of at least three independent cells stably transfected were seeded at a density of 105 cells per experiments. ml in 60-mm-diameter culture dishes coated with collagen type I (10 g͞ml, Sigma). After incubation at 37°C, the cells were with a Zeiss LSM 510 microscope by using a ϫ63 (numerical examined under a light microscope equipped with phase- contrast optics (model IX 70, Olympus) and photographed. For aperture of 1.4) Plan Neo Fluor objective. the wounding test, cells were seeded on 35-mm-diameter dishes Results precoated with collagen type I and grown to confluence in the  presence of serum. The medium was changed every 24 h. The IKK Associates with and Phosphorylates Dok1. To test whether Dok1 associates with IKK, Myc-tagged Dok1 and Flag-tagged 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 IKK (F-IKK ) were expressed in 293T cells (Fig. 1A). Approxi- eliminate nonadherent cells. The cells were then incubated at mately 0.5–1% of Dok1 in the cell lysates coprecipitated with Ϸ  37°C and examined at different times by using a Leica (Ban- 10–20% of F-IKK . The coimmunoprecipitation was specific  nockburn, IL) DM IRB microscope (objective N plan ϫ10). The because Dok1 was not precipitated from cells in which F-IKK was Ϸ area of the wound was measured in pixels by using METAMORPH not expressed (Fig. 1A). Furthermore, 0.1% of endogenous Dok1 Ϸ   software (Universal Imaging, West Chester, PA). specifically associated with 20% of IKK in F-IKK immuno- precipitates from lysates of BJAB cells constitutively expressing  Ϸ  Immunoprecipitation, GST Pull-Down Assays, and Western Blotting. F-IKK (Fig. 1B)orwith 4% of endogenous IKK in nontrans- Immunoprecipitation, GST pull-down assays, and Western blot fected BJAB cells (Fig. 1C). Thus, corrected for the efficiency of analyses were described (15). IKK immunoprecipitation, 0.5–5% of Dok1 is stably associated with IKK in 293T epithelial cells or BJAB B lymphoblasts. By Assay of Erk Activation and Kinase Assays. Cells were deprived of using polyclonal Dok1 antibody to immunoprecipitate Dok1 from serum for 24 h and then incubated with PDGF (20 ng͞ml, BJAB B lymphoblasts, Ϸ0.5% of IKK appeared to be stably Calbiochem) for indicated times. Antibodies specific for acti- Dok1-associated, after correction for the Ϸ20% efficiency of Dok1 vated Erks and total Erks were used for immunoblotting. The in precipitation (Fig. 1C); polyclonal antibody could interfere with or vitro kinase assays were described (44). inhibit intermolecular associations. Moreover, pervanadate inhibi- tion of phosphatases substantially increased Dok1 tyrosine phos- Confocal Microscopy and Immunofluorescence. Cells were seeded in phorylation and resulted in almost 2-fold more Dok1 or phospho- 35-mm glass-bottom Petri dishes coated with collagen I, and tyrosine-Dok1 association with overexpressed F-IKK in 293T cells immunofluorescence was as described (45). F-actin distribution (Fig. 1A). Interestingly, IKK overexpression decreased Dok1 was observed after incubation of cells with rhodamine B iso- tyrosine phosphorylation (Fig. 1A), which is consistent with the
thiocyanate-conjugated phalloidin (Molecular Probes) at 0.4 possibility that IKK may down-modulate Dok1 effects down- CELL BIOLOGY units͞ml for 20 min at room temperature. Samples were imaged stream of tyrosine kinases.
Lee et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17417 Downloaded by guest on September 26, 2021 Fig. 3. Dok1 is an IKK substrate. (A)IB␣,IB,IB, and Drosophila Cactus serines that are IKK-phosphorylated are compared with HuDok1 S439,S443, S446, and S450, with MuDok1, and with HuDok2. (B) HEK 293T cells were transfected with wild-type Dok1 (F-Dok1-SSSS) or various alanine substitution  Fig. 2. IKK is a Dok1 kinase. (A) HEK 293T cells were transfected with F-Dok1 mutants, with or without F-IKK . Total cell lysates were analyzed by Western  and increasing amounts of F-IKK expression vector DNAs. Cell lysates were blot using anti-Dok1 or anti-IKK antibodies. Data are representative of two analyzed by Western blotting. *, IKK-modified Dok1. (B) HEK 293T cells were or more independent experiments. transfected with F-Dok1 together with F-IKK, F-IKKKD, F-IKK␣, or F-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 IKK Phosphorylation Sites in Dok1. HuDok1 S439S443 and S446S450 Western blot. (D) HEK 293T Cells were transfected with F-Dok1 with or without and the homologous sites in murine (Mu)Dok1 and HuDok2 are F-IKK expression vector DNA. Aliquots of whole-cell lysates were treated separated by three amino acids, as are the critical serines for with Ser͞Thr phosphatase (-PPase) or tyrosine phosphatase (leukocyte IKK phosphorylation sites in IB␣, , , and Cactus (Fig. 3A). antigen-related-Tyr-PPase), and proteins were analyzed by Western blot. HuDok1 lacks a D before S ,S S ,orS , and this fact may Similar results were obtained in three independent experiments. 439 443, 446 450 be the basis for less efficient phosphorylation, relative to IB␣, , , and Cactus (Fig. 8). However, Hu and MuDok1 have a Y In transfected HEK 293T cells, overexpressed Dok1 and IKK before S450, and phosphorylation of that tyrosine may mimic D were distributed in a reticular pattern throughout the cytoplasm, orEinIB␣ and IB, respectively, and account for the with enhancement near the plasma membrane and in cytoplas- increased association of tyrosine-phosphorylated Dok1 with mic extensions, near sites of F-actin accumulation (Fig. 1D and IKK in Fig. 1A. The increased association could also be due to data not shown). Dok1 dimerization after tyrosine phosphorylation.  IKK overexpression with Dok1 resulted in a slower migrating The role of Dok1 S439S443S446S450 in IKK phosphorylation Dok1 fraction (Fig. 2A). The mobility shift occurred with native, was further evaluated by determining whether IKK overex- Flag-, or Myc- tagged Dok1 (data not shown). The fraction of pression could induce the phosphorylation of Dok1  Dok1 shift varied directly with the amount of IKK expression A439A443A446A450 (Fig. 3B). IKK overexpression resulted in vector and the amount of IKK expressed (Fig. 2A), implicating substantial Dok1 phosphorylation and slower mobility, whereas IKK in Dok1 modification. IB kinase ␣ (IKK␣) overexpres- Dok1 A439A443A446A450 was not detectably phosphorylated, and sion also induced similar Dok1 modification, whereas KD Dok1 with A substituted for any three of the four Ss was not IKK(KD) or IKK␣(KD)did not induce Dok1 modification (Fig. extensively phosphorylated (Fig. 3B). Thus, Dok1 S439S443S446,  2B). Moreover, F-IKK(KD) or M-IKK␣(KD) had dominant and S450 are critical for IKK phosphorylation. However, Dok1 negative effects on wild-type F-IKK-induced Dok1 modifica- with As substituted for the first or last two Ss was intermediate tion (data not shown). Furthermore, Yersinia YopJ, an inhibitor in the intensity of highly phosphorylated Dok1, indicating that of IKK and other mitogen-activated protein kinases (46), S439S443 and S446S450 can independently enable extensive phos- prevented IKK-induced Dok1 modification (Fig. 2C). The phorylation with reduced efficiency (Fig. 3B). These data are Dok1 mobility shift disappeared with -Ser͞Thr phosphatase most consistent with the possibility that IKK-induced Dok1 S/T treatment, but not with leukocyte antigen-related tyrosine phos- phosphorylation extends sequentially beyond S439–S450. phatase treatment (Fig. 2D). Thus, increased IKK or IKK␣ Dok1 pS443-orpS450-specific antibodies were raised in rabbits activity causes Dok1 Ser͞Thr phosphorylation. purified on phosphopeptide affinity columns and readily de- To assess whether Dok1 is a direct IKK substrate, IKK was tected phosphorylated Dok1 in extracts from 293T cells after expressed and purified from HEK 293 cells or from Sf9 insect overexpression of IKK and Dok1 (Fig. 4). As expected, phos- cells, and incubated in an in vitro kinase reaction with Escherichia phospecific antibody reactivity was not detected with overex- coli-expressed putative substrates, GST-Dok1, GST-Dok1 pression of IKK and Dok1 A439A443A446A450 (Fig. 4). Dok1 amino acids 430–481, GST-IB␣ positive control, and GST- antibody detected Dok1 and Dok1 A439A443A446A450 equally IBaA32A36 negative control proteins. IKK from 293 or Sf9 (Fig. 4). Antibodies to pS443 and pS450 reacted at low levels with cells specifically phosphorylated GST-IB␣ and, to a lesser wild-type Dok1, but not with Dok1 A439A443A446A450 in the extent, GST-Dok1, GST-Dok1 430–481, and IKK, whereas absence of cotransfected IKK (Fig. 4). Some of this low-level IKK failed to phosphorylate GST- IBaA32A36 (Fig. 8, which reactivity is likely due to phosphorylation by endogenous IKK, is published as supporting information on the PNAS web site). given the high antibody specificity for Dok1 over Dok1 Overexpression of mitogen-activated protein kinase͞Erk kinase A439A443A446A450. However, pS450 antibody did have low non- kinase 1, an upstream activator of IKK (47) also induced Dok1 phosphospecific reactivity with Dok1 A450, whereas antibody to mobility shift in 293T cells (data not shown). These data indicate pS443 appeared to lack reactivity with Dok1 A443 (Fig. 4). Overall, that IKK can directly phosphorylate Dok1 or Dok1 amino acids these data indicate that antibody to pS443 and pS450 have 430 and 481 and that Dok1 amino acids 430–481 are a sufficient substantial specificity and that IKK overexpression causes substrate for IKK phosphorylation. Dok1 phosphorylation at these sites. Similar specificity was
17418 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408061101 Lee et al. Downloaded by guest on September 26, 2021 Fig. 4. Dok1 pS443- and pS450-specific antibodies. HEK 293T cells were transfected with an expression vector for F-Dok1-SSSS, AAAA, ASSS, SASS, Fig. 5. TNF-␣, IL-1, or ␥ radiation induces Dok1 pS443 and pS450. (A) HEK 293 SSAS, or SSSA with or without F-IKK. Dok1 phosphorylation was evaluated by cells stably transfected with F-Dok1-SSSS were treated with TNF-␣ (20 ng͞ml) using anti-pS443 or anti-pS450 affinity-purified antibody. Dok1 and IKK or IL-1 (20 ng͞ml), and protein extracts were analyzed by immunoblot using levels were monitored by Western blot. These data are representative of at Dok1 and Dok1 pS443. (B) HeLa cells were treated with TNF-␣ (20 ng͞ml) for least three independent experiments. 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 obtained with antibody to peptide pS446, but not with antibody from a normal control (lymphoblastoid cell line 2145) or from two people with to peptide pS439 (data not shown). 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 TNF-␣, IL-1, and ␥ Irradiation Induce IKK-Mediated Dok1 Phosphor- data are representative of two or more independent experiments. 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 was much less than that induced by IKK or ionizing radiation TNF-␣ or IL-1, potent IKK activators. Whereas total Dok1 (data not shown). Taken together, these data indicate that TNF-, levels were similar in most clones and did not change after IL-1-, or ␥ radiation-induced IKK activation results in Dok1 TNF-␣ or IL-1 treatment, TNF-␣ or IL-1 consistently induced S443, S450 phosphorylation. pS443 and pS450 reactivity within 10 min. Phosphorylation was maximal at 10–20 min and persisted at nearly maximal levels for Dok1 S439,S443,S446, and S450 Are Required for Dok1 Effects on Cell ͞ at least 30 min (Fig. 5A and data not shown for pS450). As Proliferation, on PDGF-Induced Erk1 2 Activation, and on Cell Motility. expected, TNF-␣ or IL-1 did not cause Dok1-AAAA phosphor- Wild-type Dok1 overexpression down-modulates cell prolifera- ylation, as was evident by the absence of change in Dok1 pS443 tion and mitogen-activated protein kinase activation (16). The  or pS450 antibody reactivity in Western blots of 293 cell extracts role of the Dok1 IKK phosphorylation sites in Dok1 effects on ͞ (data not shown). TNF-␣ also induced endogenous Dok1 S443 cell proliferation and Erk1 2 kinase activity in 293 cells was and S450 phosphorylation by 10 min in HeLa cells (Fig. 5B). evaluated by comparing the effects of stable Dok1 or Dok1- Moreover, overexpression of dominant-negative IKK(KD) in- AAAA overexpression on cell proliferation and Erk1͞2 kinase hibited TNF-␣ induced Dok1 phosphorylation in 293 cells activity. As expected (16), cells overexpressing Dok1 grew more expressing F-Dok1 (data not shown). slowly than control cells (Fig. 6A). In contrast, cells expressing Ionizing radiation also activated IKK (48, 49) and induced similar amounts of Dok1-AAAA grew at a rate that was Dok1 pS450 reactivity, which reached its maximum at 1–2 h and indistinguishable from control cells (Fig. 6A). PDGF also in- persisted for at least 6 h (Fig. 5D). Radiation-induced phosphor- duced less Erk1͞2 phosphorylation in cells that overexpress ylated Dok1 had an electrophoretic mobility similar to that wild-type Dok1, whereas PDGF-induced Erk1͞2 phosphoryla- observed with IKK overexpression (data not shown). Phos- tion was normal or elevated in cells that overexpress Dok1- phorylation of S443 was also induced, but to lesser extent (data AAAA (Fig. 6B). Thus, the Dok1 serines that are phosphory- not shown). Ionizing radiation-induced IKK-mediated NF-B lated by IKK are required for Dok1 overexpression-mediated activation depends on the kinase ATM that is mutated in AT (48, inhibition of Erk1͞2 phosphorylation and cell proliferation, 49). As expected, ATM was required for Dok1 phosphorylation, which is consistent with a role for IKK-mediated phosphory- and Dok1 S450 was not phosphorylated in lymphoblastoid cell lation in Dok1 effects downstream of tyrosine kinases. lines from two AT patients (Fig. 5D; AT-11 and AT-14) har- As described (13, 18, 19), Dok1 overexpression increased 293 boring a nonfunctional truncated ATM protein (43). Thus, cell spreading and migration on coverslips coated with collagen ionizing radiation-induced Dok1 phosphorylation is ATM- I (Fig. 7A). Although polyclonal cells converted to GFP expres- dependent. Phosphorylation of p53 at S15 was observed in AT sion were indistinguishable from polyclonal cells expressing mutant cell lines at 20 Gy because other ATM family members GFP-Dok1, in their growth on plastic (except for GFP-Dok1 such as ATR can phosphorylate p53 (50). These data indicate localization to the cytoplasm and GFP distribution throughout that ATM is the principal kinase for the initiation of Dok1 the cell), cells expressing GFP-Dok1 became flatter and more phosphorylation after ionizing radiation exposure. We cannot spread out than GFP-expressing cells within hours of plating on exclude the possibility that ATM also directly phosphorylates a collagen-I substrate in complete medium with serum (Fig. 7A Dok1 because Dok1 S310 and S450 are potential SQ͞TQ ATM Left). Furthermore, GFP-Dok1-expressing cells migrated more phosphorylation motif sites (1, 50). In fact, ATM overexpression rapidly into a wound in the cell layer than did GFP control cells CELL BIOLOGY induced Dok1 pS450 reactivity, but the extent of phosphorylation (Fig. 7B Right). In contrast, Flag-Dok1-AAAA-converted cells
Lee et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17419 Downloaded by guest on September 26, 2021 Fig. 6. Dok1 inhibits cell proliferation and Erk1͞2 phosphorylation, whereas Dok1-AAAA does not affect cell proliferation and slightly increases Erk1͞2 phosphorylation. (A) HEK 293 cells stably transfected with Dok1, Dok1-AAAA, Fig. 7. Dok1, Dok1-SSEE, and Dok1-EEEE increase cell migration, whereas ͞ ͞ or empty pcDNA3 expression vectors were monitored for growth. Data are Dok1-AAAA inhibits cell migration. (A) Polyclonal selected 293 GFP-or 293 mean Ϯ SE of three independent experiments carried out in triplicate. Similar GFP-Dok1-expressing cells were plated in serum on coverslips coated with results were also obtained from two other independent sets of expressing collagen I. Cells were observed by fluorescence (FL) and phase contrast (Ph) for clones. (B) Equal protein extracts from PDGF-treated pcDNA3-, Dok1-, or wound healing (WH) 2 h later. Healing (WH) in the cell layer was photo- Dok1-AAAA-transfected cells lines were analyzed by immunoblotting for graphed for the indicated cell lines at 0 (WH 0) and 2 h (WH 2). Cell migration pERK1͞2, Erk1͞2, and Dok1. Data are representative of at least three inde- was evaluated based on cell filling of the wound area by using METAMORPH pendent experiments. 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 is plotted. Data are means Ϯ SE of three independent experiments carried out were significantly slower in migration than Flag-Dok1 cells (Fig. in triplicate. 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 S ,S ,S , The data presented here support the hypothesis that Dok1 439 443 446  and S450 in Dok1 effects on cell spreading and migration, which S439,S443,S446, and S450, which are phosphorylated by IKK , are is consistent with previously observed TNF-␣- or IL-1-induced critical for previously characterized Dok1 effects downstream of  increase in cell spreading; such effects have also been attributed tyrosine kinase signaling. Activated IKK phosphorylation of to other mitogen-activated protein kinase pathways (51–53). these Dok1 serines may augment Dok1 effects. Indeed, mutation Importantly, Dok1 SSSS or AAAA overexpression did not of Dok1 S439,S443,S446, and S450 to A resulted in Dok1 being ͞ affect IKK-mediated IB␣ phosphorylation or subsequent unable to inhibit PDGF-mediated Erk1 2 activation or cell degradation after TNF-␣ treatment (data not shown), indicating proliferation. that Dok1 does not compete with IB␣ for IKK, even when Most relevant to a role for Dok1 serine phosphorylation were motility studies in which wild-type Dok1 overexpression in- Dok1 is overexpressed. creased cell motility, Dok1 with S439,S443,S446, and S450 mutated Discussion to A439,A443,A446, and A450 was unable to increase cell motility, and Dok1 with S ,S ,S , and S mutated to E ,E , These experiments indicate that physiologic IKK activation 439 443 446 450 439 443 E , and E was slightly superior to wild-type Dok1 in increas- results in Dok1 S ,S , and S phosphorylation. IKK (and 446 450 443 446 450 ing cell motility. Moreover, as shown in Fig. 1D, Dok1 and IKK ␣) associated with and phosphorylated Dok1 on S439S443 and  had a diffuse reticular distribution, with accentuation at sites of S446S450. Extensive IKK -mediated Dok1 phosphorylation, in F-actin, which is consistent with a role for IKK in filopodia vivo, depended on S439,S443,S446, and S450, and Dok1 amino acids  formation, possibly related to cell motility. Altogether, these 430–481 were phosphorylated by recombinant IKK , in vitro. biochemical and reverse genetic data are consistent with the ␣  Furthermore, TNF- , IL-1, or ionizing radiation induced IKK hypothesis that IKK phosphorylation of Dok1 is important for activation and Dok1 S443,S446, and S450 phosphorylation, in vivo, Dok1 effects on cell motility. as assayed with Dok1 pS site-specific antisera. Moreover, Dok1 Other evidence supports a role for Dok1 in cell motility. phosphorylation after ionizing radiation was ATM-dependent, Mu-Dok1 Y361, which is equivalent to Hu-Dok1 Y362, is also a which was consistent with the expected ATM role in activating specific c-Abl substrate in fibroblasts and mediates Nck recruit-  IKK . Dok1 S443,S446, and S450 phosphorylations were down- ment followed by Pak1 or mitogen-activated protein kinase Erk stream of TNF-␣, IL-1, or ionizing radiation-induced IKK kinase kinase 1 activation (13, 54–57). Mouse fibroblasts lacking activation in human epithelial and B cells. c-Abl, Dok1, or Nck have fewer filopodia than cells expressing Dok1 was a less efficient than IB␣ as an in vitro substrate for the disrupted gene (57). activated IKK. Furthermore, Dok1 overexpression did not Dok1 is now among a growing list of ‘‘nonclassical’’ IKK  affect IKK -mediated NF- B activation. Dok1 therefore ap- substrates. Dok1 S439S443 and S446S450 are similar in amino acid pears to be an alternative IKK substrate that does not inhibit sequence to IB␣ S32S36. Furthermore, Dok1 Y449 is an impor- IKK phosphorylation of IB␣. tant Csk tyrosine kinase site and Y449 phosphorylation next to
17420 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408061101 Lee et al. Downloaded by guest on September 26, 2021 S450 may mimic the D or E that precedes S32 and S36 in IB␣. knocked down, or IKK chemically inhibited cells. Investigations  Y449 phosphorylation would enhance Dok1 similarity to classical with murine IKK knock out fibroblasts were hindered by poor IKK substrates. Y449 phosphorylation also increases Dok1, reactivity of HuDok1 pS-specific sera with MuDok1. We also SH2D1A, SHP-2, or SHIP binding (11–15). Whereas most IKK note that IKK␣ was present in Dok1 immunoprecipitations and substrates, including IB␣, p65, or p105 are important for NF-B could also have a role in Dok1 S phosphorylation. activation (26), -catenin, SRC-3, and insulin receptor substrate ␣ 1, another Dok family member, have also been identified to be We thank T. Gilmore (Boston University, Boston) for GST I B   plasmids; G. Mosialos, T. Gilmore, E. Cahir-McFarland, J. Hall, nonclassical IKK substrates (58–60). These other IKK sub- M. Tommasino, F. Roy and Z.-Q. Wang for contributing advice; strates are also phosphorylated by other kinases, such as glyco- J. Cheney for editing; G. Mollon (International Agency for Research gen synthetase kinase 3 (61) and Dok1 may also be a more on Cancer) for illustrations; D. Goeddel, H. Allen, J. Dixon, Y. Shiloh, promiscuous Ser͞Thr kinase substrate. However, we have re- M. Kastan, G. Johnson, J. Woodgett, J. Cambier, and G. Lenoir for cently found that glycogen synthetase kinase 3 overexpression reagents; I. Verma (The Salk Institute, La Jolla, CA) for IKK null in 293T cells had less effect than IKK on Dok1 phosphoryla- mouse embryonic fibroblasts; and B. Chapot for technical assistance. tion, by using Dok1 pS450-specific antibody (data not shown). This work was supported by National Institutes of Health Grants  ␣ CA47006 and CA85180 (to E.D.K.), by a fellowship from the Asso- Further evaluation of the role of IKK in mediating TNF- , ciation pour la Recherche sur le Cancer (to B.S.), and by Ministe`re de IL-1, or ␥ radiation effects through phosphorylation of Dok1 l’Education Nationale de la Recherche et de la Technologie fellow- S439–450 should include studies in IKK knockout, RNAi ships (to F.S. and O.D.).
1. Carpino, N., Wisniewski, D., Strife, A., Marshak, D., Kobayashi, R., Stillman, 34. Saijo, K., Mecklenbrauker, I., Santana, A., Leitger, M., Schmedt, C. & B. & Clarkson, B. (1997) Cell 88, 197–204. Tarakhovsky, A. (2002) J. Exp. Med. 195, 1647–1652. 2. Yamanashi, Y. & Baltimore, D. (1997) Cell 88, 205–211. 35. Su, T. T., Guo, B., Kawakami, Y., Sommer, K., Chae, K., Humphries, L. A., 3. Cong, F., Yuan, B. & Goff, S. P. (1999) Mol. Cell. Biol. 19, 8314–8325. Kato, R. M., Kang, S., Patrone, L., Wall, R., et al. (2002) Nat. Immunol. 3, 4. Di Cristofano, A., Carpino, N., Dunant, N., Friedland, G., Kobayashi, R., Strife, 780–786. A., Wisniewski, D., Clarkson, B., Pandolfi, P. P. & Resh, M. D. (1998) J. Biol. 36. Wooten, M. W., Seibenhener, M. L., Neidigh, K. B. & Vandenplas, M. L. (2000) Chem. 273, 4827–4830. Mol. Cell. Biol. 20, 4494–4504. 5. Grimm, J., Sachs, M., Britsch, S., Di Cesare, S., Schwarz-Romond, T., Alitalo, 37. Yeung, K. C., Rose, D. W., Dhillon, A. S., Yaros, D., Gustafsson, M., K. & Birchmeier, W. (2001) J. Cell Biol. 154, 345–354. Chatterjee, D., McFerran, B., Wyche, J., Kolch, W. & Sedivy, J. M. (2001) Mol. 6. Lemay, S., Davidson, D., Latour, S. & Veillette, A. (2000) Mol. Cell. Biol. 20, Cell. Biol. 21, 7207–7217. 2743–2754. 38. Herndon, T. M., Shan, X. C., Tsokos, G. C. & Wange, R. L. (2001) J. Immunol. 7. Nelms, K., Snow, A. J., Hu-Li, J. & Paul, W. E. (1998) Immunity 9, 13–24. 166, 5654–5664. 8. White, M. F. (1998) Mol. Cell. Biochem. 182, 3–11. 39. Khan, W. N. (2001) Immunol. Res. 23, 147–156. 9. Songyang, Z., Yamanashi, Y., Liu, D. & Baltimore, D. (2001) J. Biol. Chem. 40. Weil, R., Schwamborn, K., Alcover, A., Bessia, C., Di Bartolo, V. & Israel, A. 276, 2459–2465. (2003) Immunity 18, 13–26. 10. Zhao, M., Schmitz, A. A., Qin, Y., Di Cristofano, A., Pandolfi, P. P. & Van 41. Yoshida, K., Yamashita, Y., Miyazato, A., Ohya, K., Kitanaka, A., Ikeda, U., Aelst, L. (2001) J. Exp. Med. 194, 265–274. Shimada, K., Yamanaka, T., Ozawa, K. & Mano, H. (2000) J. Biol. Chem. 275, 11. Bhat, A., Johnson, K. J., Oda, T., Corbin, A. S. & Druker, B. J. (1998) J. Biol. 24945–24952. Chem. 273, 32360–32368. 42. Sylla, B. S., Hung, S. C., Davidson, D. M., Hatzivassiliou, E., Malinin, N. L., 12. Neet, K. & Hunter, T. (1995) Mol. Cell. Biol. 15, 4908–4920. Wallach, D., Gilmore, T. D., Kieff, E. & Mosialos, G. (1998) Proc. Natl. Acad. 13. Noguchi, T., Matozaki, T., Inagaki, K., Tsuda, M., Fukunaga, K., Kitamura, Y., Sci. USA 95, 10106–10111. Kitamura, T., Shii, K., Yamanashi, Y. & Kasuga, M. (1999) EMBO J. 18, 43. Ange`le, S., Lauge´, A., Fernet, M., Moullan, N., Beauvais, P., Couturier, P., 1748–1760. Stoppa-Lyonnet, D. & Hall, J. (2003) Hum. Mutat. 21, 169–170. 14. Sattler, M., Verma, S., Pride, Y. B., Salgia, R., Rohrschneider, L. R. & Griffin, 44. Li, N. & Karin, M. (2000) Methods Enzymol. 319, 273–279. J. D. (2001) J. Biol. Chem. 276, 2451–2458. 45. Ory, S., Munari-Silem, Y., Fort, P. & Jurdic, P. (2000) J. Cell Sci. 113, 15. Sylla, B. S., Murphy, K., Cahir-McFarland, E., Lane, W. S., Mosialos, G. & 1177–1188. Kieff, E. (2000) Proc. Natl. Acad. Sci. USA 97, 7470–7475. 46. Orth, K., Palmer, L. E., Bao, Z. Q., Stewart, S., Rudolph, A. E., Bliska, J. B. 16. Di Cristofano, A., Niki, M., Zhao, M., Karnell, F. G., Clarkson, B., Pear, W. S., & Dixon, J. E. (1999) Science 285, 1920–1922. Van Aelst, L. & Pandolfi, P. P. (2001) J. Exp. Med. 194, 275–284. 47. Lee, F. S., Peters, L. C., Dang, L. C. & Maniatis, T. (1998) Proc. Natl. Acad. 17. Wick, M. J., Dong, L. Q., Hu, D., Langlais, P. & Liu, F. (2001) J. Biol. Chem. Sci. USA 95, 9319–9324. 276, 42843–42850. 48. Li, N., Banin, S., Ouyang, H., Li, G., Courtois, G., Shiloh, Y., Karin, M. & 18. Yamanashi, Y., Tamura, T., Kanamori, T., Yamane, H., Nariuchi, H., Rotman, G. (2001) J. Biol. Chem. 276, 8898–8903. Yamamoto, T. & Baltimore, D. (2000) Genes Dev. 14, 11–16. 49. Piret, B., Schoonbroodt, S. & Piette, J. (1998) Oncogene 18, 2261–2271. 19. Hosooka, T., Noguchi, T., Nagai, H., Horikawa, T., Matozaki, T., Ichihashi, M. 50. Kastan, M. B. & Lim, D. (2000) Nat. Rev. Mol. Cell Biol. 1, 179–186. & Kasuga, M. (2001) Mol. Cell. Biol. 21, 5437–5446. 51. Chen, Y., Ke, Q., Yang, Y., Rana, J. S., Tang, J., Morgan, J. P. & Xiao, Y. F. 20. Yamakawa, N., Tsuchida, K. & Sugino, H. (2002) EMBO J. 21, 1684–1694. (2003) FASEB J. 17, 2231–2239. 21. Kato, I., Takai, T. & Kudo, A. (2002) J. Immunol. 168, 629–634. 52. Corcione, A., Ottonello, L., Tortolina, G., Tasso, P., Ghiotto, F., Airoldi, I., 22. Martelli, M. P., Boomer, J., Bu, M. & Bierer, B. E. (2001) J. Biol. Chem. 276, Taborelli, G., Malavasi, F., Dallegri, F. & Pistoia, V. (1997) Blood 90, 45654–45661. 4493–4501. 23. Nemorin, J. G., Laporte, P., Berube, G. & Duplay, P. (2001) J. Immunol. 166, 53. Miossec, P., Yu, C. L. & Ziff, M. (1984) J. Immunol. 133, 2007–2011. 4408–4415. 54. Master, Z., Jones, N., Tran, J., Jones, J., Kerbel, R. S. & Dumont, D. J. (2001) 24. Tamir, I., Stolpa, J. C., Helgason, C. D., Nakamura, K., Bruhns, P., Daeron, M. EMBO J. 20, 5919–5928. & Cambier, J. C. (2000) Immunity 12, 347–358. 55. Murakami, H., Yamamura, Y., Shimono, Y., Kawai, K., Kurokawa, K. & 25. Liang, X., Wisniewski, D., Strife, A., Clarkson, B. & Resh, M. D. (2002) J. Biol. Takahashi, M. (2002) J. Biol. Chem. 277, 32781–32790. Chem. 277, 13732–13738. 56. Yujiri, T., Ware, M., Widmann, C., Oyer, R., Russell, D., Chan, E., Zaitsu, Y., 26. Karin, M. & Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621–663. Clarke, P., Tyler, K., Oka, Y., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 27. Karin, M. & Lin, A. (2002) Nat. Immunol. 3, 221–227. 7272–7277. 28. Silverman, N. & Maniatis, T. (2001) Genes Dev. 15, 2321–2342. 57. Woodring, P. J., Meisenhelder, J., Johnson, S.A., Zhou, G. L., Field, J., Shah, 29. Baldwin, A. S. J. (1996) Annu. Rev. Immunol. 14, 649–683. K., Bladt, F., Pawson, T., Niki, M., Pandolfi, P. P., et al. 2004) J. Cell Biol. 165, 30. Arsura, M., Mercurio, F., Oliver, A. L., Thorgeirsson, S. S. & Sonenshein, G. E. 493–503. (2000) Mol. Cell. Biol. 20, 5381–5391. 58. Gao, Z., Hwang, D., Bataille, F., Lefevre, M., York, D., Quon, M. J. & Ye, J. 31. Baumann, B., Weber, C. K., Troppmair, J., Whiteside, S., Israel, A., Rapp, (2002) J. Biol. Chem. 277, 48115–48121. U. R. & Wirth, T. (2000) Proc. Natl. Acad. Sci. USA 97, 4615–4620. 59. Lamberti, C., Lin, K. M., Yamamoto, Y., Verma, U., Verma, I. M., Byers, S. 32. Kane, L. P., Mollenauer, M. N., Xu, Z., Turck, C. W. & Weiss, A. (2002) Mol. & Gaynor, R. B. (2001) J. Biol. Chem. 276, 42276–42286. Cell. Biol. 22, 5962–5974. 60. Wu, R. C., Qin, J., Hashimoto, Y., Wong, J., Xu, J., Tsai, S. Y., Tsai, M. J. &
33. Petro, J. B., Rahman, S. M., Ballard, D. W. & Khan, W. N. (2000) J. Exp. Med. O’Malley, B. W. (2002) Mol. Cell. Biol. 22, 3549–3561. CELL BIOLOGY 191, 1745–1753. 61. Harwood, A. J. (2001) Cell 105, 821–824.
Lee et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17421 Downloaded by guest on September 26, 2021