NF-kappaB activation in T cells requires discrete control of IkappaB kinase alpha/beta (IKKalpha/beta) phosphorylation and IKKgamma ubiquitination by the ADAP adapter protein

Abstract

NF-kappaB activation following engagement of the antigen-specific T cell receptor involves protein kinase C-theta-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IkappaB kinase (IKK). We previously identified a novel role for the adhesion- and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. In this study, we identify a novel site in ADAP that is critical for association with the TAK1 kinase. ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to protein kinase C-theta. ADAP is not required for TAK1 activation. Although both the TAK1 and the CARMA1 binding sites in ADAP are essential for IkappaB alpha phosphorylation and degradation and NF-kappaB nuclear translocation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKgamma. Thus, distinct sites within ADAP control two key activation responses that are required for NF-kappaB activation in T cells.

FIGURE 1.

ADAP regulates TAK1 recruitment to the PKCθ signalosome. A, T cells from control ADAP^+/− mice (Ctrl) and ADAP^−/− mice were left unstimulated or CD3/CD28-stimulated for the indicated times (in min). Cells were lysed and probed for phospho-IKK (p-IKK) and IKKα/β. B–E, control and ADAP^−/− T cells were left unstimulated (U or −) or stimulated with anti-CD3/CD28 (3/28 or +) or phorbol 12-myristate 13-acetate (P) for 15 min and lysed. B, PKCθ IPs were probed for IKKα/β and PKCθ. C, PKCθ IPs were probed for TAK1, ADAP, CARMA1, and PKCθ. D, TAK1 IPs were probed for ADAP, PKCθ, CARMA1, and TAK1. E, control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were first transduced with control adenovirus encoding Thy1.1 alone (Thy) or wild-type ADAP (WT) and cultured for 3 days prior to CD3/CD28 stimulation. PKCθ IPs were probed for ADAP, TAK1, CARMA1, and PKCθ.

FIGURE 2.

A site in the C-terminal end of ADAP is critical for TAK1 association. A, schematic representation of the CARMA1 binding ADAP mutant (ADAP ΔCAR) and TAK1 binding ADAP mutant (ADAP ΔTAK). PRO, proline-rich region, E/K, glutamic and lysine-rich region; hSH3, non-canonical helical SH3 domain. B, Jurkat T cells were transfected with the indicated HA-tagged ADAP constructs and stimulated with anti-CD3 monoclonal antibody OKT3 for 15 min. Anti-HA IPs were probed for HA-ADAP, TAK1, and BCL10. C, control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were transduced with adenovirus encoding Thy1.1 alone (Thy) or wild-type ADAP (WT), the ADAP ΔCAR mutant, or the ADAP ΔTAK mutant prior to CD3/CD28 stimulation for 15 min. ADAP IPs were probed for TAK1, CARMA1, BCL-10, and ADAP. D, control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were transduced and stimulated as in C. PKCθ IPs were probed for ADAP, TAK1, BCL10, CARMA1, and PKCθ.

FIGURE 3.

Independent control of IKKγ ubiquitination and IKKα/β phosphorylation by ADAP. Control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were transduced with adenovirus encoding Thy1.1 alone (Thy) or wild-type ADAP (WT), the ADAP ΔCAR mutant, or the ADAP ΔTAK mutant prior to CD3/CD28 stimulation for 15 min (A and C) or for 15 or 45 min (B). A, IKKγ IPs were probed for ubiquitin and IKKγ. B, IKKα/β IPs were probed for phosphorylated IKK and IKK. C, in vitro kinase assays were performed with TAK1 IPs. Phosphorylation of GST·IKK was assessed by Western blotting with an anti-phospho-IKK antibody (p-GST IKK). Samples were also probed with an anti-IKK antibody (GST IKK). D, flow cytometry analysis of T cells infected with adenovirus with an anti-Thy1.1 antibody, which detects the Thy1.1 cell surface protein expressed by all recombinant adenoviruses used in this study. Cell lysates were also immunoblotted with an anti-ADAP antibody to confirm ADAP expression (bottom).

FIGURE 4.

The ADAP ΔCAR and ADAP ΔTAK mutants are each unable to restore CD3/CD28-mediated IkBα phosphorylation and degradation and p65 nuclear translocation. Control hCAR⁺ T cells (Ctrl) and hCAR⁺ ADAP^−/− T cells were transduced with adenovirus encoding Thy1.1 alone (Thy) or wild-type ADAP (WT), the ADAP ΔCAR mutant, or the ADAP ΔTAK mutant prior to CD3/CD28 stimulation for 15 or 45 min. A, lysates were immunoblotted with antibodies specific for IkBα, phospho-IκB (p-IκB), phospho-Erk (p-ERK), and Erk. B, nuclear and cytoplasmic extracts were analyzed by immunoblotting with antibodies specific for p65 and the nuclear marker lamin A/C. Cytoplasmic extracts were also probed with an anti-ADAP antibody to verify expression of ADAP.