IκB kinase β phosphorylates Dok1 serines in response to TNF, IL-1, or γ radiation

doi:10.1073/pnas.0408061101

IκB kinase β phosphorylates Dok1 serines in response to TNF, IL-1, or γ radiation

^*International Agency for Research on Cancer, 69008 Lyon, France; ^¶Ecole Normale Supérieure, 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

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 IκB kinase β (IKKβ) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKβ phosphorylation of Dok1 depended on Dok1 S₄₃₉, S₄₄₃, S₄₄₆, and S₄₅₀. Recombinant IKKβ also phosphorylated Dok1 or Dok1 amino acids 430–481 in vitro. TNF-α, IL-1, γ radiation, or IKKβ overexpression phosphorylated Dok1 S₄₄₃, S₄₄₆, and S₄₅₀ in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S₄₃₉, S₄₄₃, S₄₄₆, and S₄₅₀ mutated to A was not phosphorylated by IKKβ in vivo. Surprisingly, mutant Dok1 A₄₃₉, A₄₄₃, A₄₄₆, and A₄₅₀ 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 A₄₃₉, 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 IKKβ phosphorylates Dok1 S₄₃₉S₄₄₃ and S₄₄₆S₄₅₀ after TNF-α, IL-1, or γ-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.

Footnotes

↵ ¶¶ To whom correspondence should be addressed at: International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008 Lyon, France. E-mail: sylla{at}iarc.fr.
↵ † Present address: Beth Israel Hospital, Harvard Medical School, Boston, MA 02115.
↵ ‡ Present address: Institut National de la Santé et de la Recherche Médicale Unit 450, Institut Biomédical des Cordeliers, 75006 Paris, France.
↵ § C.A., F.S., and O.D. contributed equally to this work.
↵ ∥ Present address: Centre d'Oncologie Génétique, Centre Léon Bérard, 69008 Lyon, France.
↵ ** Present address: Complexe Universitaire des Céseaux, 63174 Aubière, France.
↵ †† Present address: Schering–Plough Laboratory for Immunological Research, 69571 Dardilly, France.
Author contributions: S.L., C.A., F.S., O.D., P.J., E.D.K., and B.S.S. designed research; S.L., C.A., F.S., O.D., J.A., V.P., J.M., B.S., and B.S.S. performed research; S.L., C.A., F.S., O.D., J.A., V.P., J.M., B.S., R.K., P.J., E.D.K., and B.S.S. contributed new reagents/analytic tools; S.L., C.A., F.S., O.D., J.A., V.P., J.M., B.S., R.K., P.J., E.D.K., and B.S.S. analyzed data; and S.L., C.A., F.S., O.D., P.J., E.D.K., and B.S.S. wrote the paper.
Abbreviations: IKKβ, IκB kinase β; IKKα, IκB kinase α; 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, human embryonic kidney; F-IKKβ, Flag-tagged IKKβ.