ADAP regulates cell cycle progression of T cells via control of cyclin E and Cdk2 expression through two distinct CARMA1-dependent signaling pathways

Abstract

Adhesion and degranulation-promoting adapter protein (ADAP) is a multifunctional scaffold that regulates T cell receptor-mediated activation of integrins via association with the SKAP55 adapter and the NF-κB pathway through interactions with both the CARMA1 adapter and serine/threonine kinase transforming growth factor β-activated kinase 1 (TAK1). ADAP-deficient T cells exhibit impaired proliferation following T cell receptor stimulation, but the contribution of these distinct functions of ADAP to this defect is not known. We demonstrate that loss of ADAP results in a G₁-S transition block in cell cycle progression following T cell activation due to impaired accumulation of cyclin-dependent kinase 2 (Cdk2) and cyclin E. The CARMA1-binding site in ADAP is critical for mitogen-activated protein (MAP) kinase kinase 7 (MKK7) phosphorylation and recruitment to the protein kinase C θ (PKCθ) signalosome and subsequent c-Jun kinase (JNK)-mediated Cdk2 induction. Cyclin E expression following T cell receptor stimulation of ADAP-deficient T cells is transient and associated with enhanced cyclin E ubiquitination. Both the CARMA1- and TAK1-binding sites in ADAP are critical for restraining cyclin E ubiquitination and turnover independently of ADAP-dependent JNK activation. T cell receptor-mediated proliferation was most dramatically impaired by the loss of ADAP interactions with CARMA1 or TAK1 rather than SKAP55. Thus, ADAP coordinates distinct CARMA1-dependent control of key cell cycle proteins in T cells.

Fig 1

Impaired proliferation and block in G₁-S transition in ADAP^−/− T cells upon CD3/CD28 stimulation. Purified naïve hCAR control (Ctrl) or hCAR/ADAP^−/− T cells were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP). (A) Cells were stained with anti-Thy1.1-APC and analyzed by flow cytometry. (B) Cell lysates were prepared, and Western blotting was performed with anti-ADAP antibody. (C) Cells were labeled with CFSE and stimulated with anti-CD3 and anti-CD28 antibodies for 42 h prior to flow cytometry analysis. (D) Cell cycle analysis was performed on the transduced T cells using DAPI staining. Results for both the proliferation and cell cycle analysis are representative of at least five independent experiments.

Fig 2

Impaired proliferation and block in G₁-S transition in ADAP^−/− T cells upon antigen challenge in vivo. (A) Naïve DO11.10 hCAR control (Ctrl) or DO11.10 hCAR/ADAP^−/− T cells were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP), stained with CFSE, and then transferred into BALB/c mice. Two hours after T cell transfer, host mice were injected with OVAp and spleens were harvested 42 h after OVAp injection. T cells expressing Thy1.1 and the DO11.10 T cell receptor were identified by flow cytometry and analyzed for CFSE dye dilution. (B) CD4 T cells transduced and transferred as described for panel A were analyzed for cell cycle transition using DAPI staining. Results for both the proliferation and cell cycle analysis are representative of at least three independent experiments.

Fig 3

ADAP is critical for efficient cyclin E and Cdk2 accumulation. (A and B) Purified naïve control (Ctrl) or ADAP^−/− CD4 T cells were activated with anti-CD3 and anti-CD28 antibodies for the indicated times, and cell extracts were prepared for qRT-PCR or Western blotting. (A) qRT-PCR showing fold change relative to β-actin for the early activation marker CD69, cyclin E, and Cdk2. (B) Total cell lysates from the indicated time course performed in parallel with the experiments whose results are shown in panel A were subjected to Western blotting with antibodies to cyclin E, Cdk2, and β-actin. (C) Wild-type or ADAP^−/− T cells were activated with anti-CD3 and anti-CD28 antibodies for 24 h. Cyclin E immunoprecipitates or control IgG were prepared, and Western blots were probed for cyclin E and ubiquitin. IP, immunoprecipitation; KO, knockout. Results are representative of two independent experiments performed.

Fig 4

ADAP interaction with CARMA1 and TAK1 is critical for expression and maintenance of Cdk2 and cyclin E. (A) Naïve hCAR control type (Ctrl) or hCAR ADAP^−/− T cells (ADAP^−/−) were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP) and then stimulated with anti-CD3 and anti-CD28 antibodies for 48 h. Lysates were probed for cyclin E (Cyc E), phospho-Cdk2 (p-cdk2), Cdk2, cyclin D1 (Cyc D1), cyclin D2 (Cyc D2), and Cdk4. (B) Densitometry values for cyclin E and Cdk2 from three independent experiments performed as described for panel A were obtained using Odyssey software. Values were normalized to the control wild-type unstimulated level and are expressed as arbitrary units (A.U.). (C) Cells transduced and activated as described for panel A were harvested at 24 h and immunoprecipitated for cyclin E, and Western blots were performed for cyclin E and ubiquitin. Similar results were obtained in two independent experiments.

Fig 5

The CARMA1-binding site in ADAP is critical for TCR-dependent JNK activation and induction of Cdk2. (A) Control (Ctrl), CARMA^−/−, and ADAP^−/− T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 48 h, lysed, and analyzed by Western blotting with antibodies specific for JNK, phospho-JNK, cyclin E, Cdk2, and β-actin. (B) Naïve hCAR control or hCAR/ADAP^−/− T cells were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP) and then stimulated with anti-CD3 and anti-CD28 antibodies. Lysates were probed by Western blotting with anti-phospho-JNK (p-JNK) and anti-JNK antibodies. (C) Naïve hCAR control or hCAR/ADAP^−/− T cells were transduced with the indicated adenoviruses as described for panel B and stimulated with anti-CD3 and anti-CD28 antibodies for 48 h in the presence (+SP) or absence (−SP) of the JNK inhibitor SP6000125 (30 μM). Cells were harvested, lysed, and probed by Western blotting with anti-phospho-c-Jun, anti-c-Jun, anti-cyclin E, and anti-Cdk2 antibodies. Similar results were observed in at least three independent experiments.

Fig 6

The CARMA1-binding site in ADAP regulates TCR-mediated activation of MKK7. (A and B) Naïve control (Ctrl) and ADAP^−/− T cells were stimulated for the indicated time points (min) with anti-CD3 and anti-CD28 antibodies. Cell lysates were analyzed by Western blotting with antibodies specific for JNK and phospho-JNK (A) or MKK7, MKK4, phospho-MKK7 (p-MKK7), and phospho-MKK4 (B). (C) Naïve control, CARMA1^−/−, and ADAP^−/− T cells were stimulated as described for panels A and B. Cell lysates were analyzed by Western blotting with antibodies specific for MKK7 and phospho-MKK7. (D) Naïve hCAR control or hCAR/ADAP^−/− T cells were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP) and then stimulated with anti-CD3 and anti-CD28 antibodies for 20 min. Cell lysates were analyzed by Western blotting with antibodies specific for phospho-MKK7, phospho-MKK4, and phospho-ERK (p-ERK). Similar results were obtained in at least three independent experiments.

Fig 7

TCR-inducible MKK7 recruitment is dependent on the CARMA1-binding site in ADAP. (A) Naïve control (Ctrl) and ADAP^−/− T cells were stimulated for the indicated time points (min) with anti-CD3 and anti-CD28 antibodies. PKCθ immunoprecipitates were analyzed by Western blotting with antibodies specific for MKK4, MKK7, and PKCθ. (B) Naïve hCAR control or hCAR/ADAP^−/− T cells were transduced with control adenovirus expressing Thy1.1 (Thy) or adenovirus expressing Thy1.1 and wild-type ADAP (WT), ADAPΔCAR mutant (ΔCAR), ADAPΔTAK mutant (ΔTAK), or ADAPΔSKAP mutant (ΔSKAP) and then stimulated with anti-CD3 and anti-CD28 antibodies for 20 min. Cell lysates were subjected to immunoprecipitation with either an anti-PKCθ antibody or an anti-Bcl10 antibody and then analyzed by Western blotting with antibodies specific for MKK7, MKK4, PKCθ, TAK1, JNK, or Bcl10. Results are representative of three independent experiments performed.