Article Figures & Data
Figures
- FIG. 1.
CARMA1-deficient JPM 50.6 Jurkat cells fail to activate NF-κB in response to superantigen. JPM 50.6 (JPM) and parental Jurkat T cells were transfected with an NFAT/AP-1 or NF-κB luciferase (Lucif.) reporter. The next day, cells were stimulated with Daudi B cells plus SEE, and luciferase activity was determined. Results displayed are the average relative light units from three values from a single experiment, which was representative of three experiments that were performed.
- FIG. 2.
NF-κB activation by Akt is impaired in the absence of CARMA1. (A) Jurkat and JPM 50.6 cells were transfected with an NF-κB luciferase reporter and the indicated plasmids. Cells were stimulated the next day for 6 hours, and luciferase activity was determined. Open bars indicate samples stimulated with PMA alone, and filled bars indicate samples stimulated with PMA plus anti-CD28. Results are displayed as the average fold stimulation over unstimulated samples, from duplicate samples of a single experiment, representative of 10 experiments that were performed. The Western blot insert shows expression of HA-Akt (indicated by the arrow) in the transfections assayed for luciferase activity. V, empty vector. (B) Jurkat and JPM 50.6 cells were transfected with a Flag-tagged IKKβ construct, along with empty vector or Akt. The next day, cells were left unstimulated (−) or treated with 10 ng/ml PMA for 30 min. IKK-β was immunoprecipitated from cell lysates and incubated with recombinant glutathione S-transferase (GST)-IκB substrate. Phosphorylated substrate was revealed by blotting with a phospho-IκB antibody. This experiment is representative of three experiments that were performed.
- FIG. 3.
Normal Akt and GSK-3 phosphorylation in JPM 50.6 cells. Jurkat and JPM 50.6 cells were stimulated with anti-TCR and anti-CD28 antibodies for the indicated times (in minutes). Lysates from one million cells per lane were subjected to SDS-PAGE and Western blotting. Blots were probed with phospho-specific antibodies to Akt or GSK-3, stripped, and reprobed with a monoclonal antibody specific for beta-actin. All blots are from a single experiment, representative of three experiments that were performed.
- FIG. 4.
A selective Akt inhibitor partially impairs CD3/CD28-mediated NF-κB activation. (A) D10 T cells were preincubated for 30 min at room temperature without or with 20 μM Akti 1/2. Cells were then stimulated at 37°C for the indicated times (0, 15, and 30 min) with biotinylated anti-CD3/CD28 antibodies (1 μg/ml each plus 5 μg/ml streptavidin). Western blots of cell lysates were probed with a polyclonal antibody specific for phospho-serine 473 in Akt. (B) D10 T cells were pretreated with Akti 1/2 and stimulated as described above for panel A. Western blots of cell lysates were probed with a polyclonal antibody specific for IκB-α. (C) D10 T cells were transfected with an NF-κB-luciferase reporter and stimulated under the conditions indicated. PMA was used at 10 ng/ml, and Akti 1/2 was used at 20 μM. Results in all cases are representative of three experiments. Values are the average number of light units ± standard deviations (error bars) from three values of a representative experiment. The inhibition of CD3/CD28-mediated NF-κB was determined to be significant by a Student's t test analysis (P < 0.05).
- FIG. 5.
Association between Akt and CARMA1. HEK 293T cells were transfected with Myc-tagged CARMA1 and the indicated constructs. The next day, IPs were performed with anti-Myc antibody and immunoprecipitated proteins were separated by SDS-PAGE. Western blots were probed with anti-HA (A) or anti-GFP (B) antibody to detect Akt in the immunoprecipitates (IP's) or cell lysates. Blots were stripped and reprobed with anti-Myc antibody to control for levels of Myc-CARMA in the immunoprecipitates (Anti-Myc IP's). Results are representative of three (A) or five (B) experiments that were performed. Pro Mut, proline mutation.
- FIG. 6.
CARMA1 localization during T-cell activation by APCs. D10 T cells were transfected with the indicated GFP constructs and Myc-tagged CARMA1. The next day T cells—alone (A) or in conjugates (B to D) with antigen-loaded APCs—were settled onto slides. Slides were stained with Cy3-conjugated anti-Myc antibody and Alexa 647-conjugated cholera toxin B subunit to reveal lipid rafts and analyzed by confocal microscopy. (C) T cells were pretreated for 1 hour with 20 μM LY294002 before formation of conjugates. The fields in row B include a cell expressing PH-GFP but not Myc-CARMA1 (lower right). Images are representative of 20 to 30 cells analyzed in each case. The asterisks indicate the location of the APC in each series of conjugates. Unconjug., unconjugated; Akt C term-GFP, GFP fusion containing the C-terminal domain of Akt (Akt C term-GFP).
- FIG. 7.
Akt-dependent, but CARMA1-independent, phosphorylation of Bcl10. (A) Human embryonic kidney (HEK) 293T cells were transfected with Bcl10 and the plasmids indicated above the lanes. Cytoplasmic lysates were separated by SDS-PAGE and Western blotted with an antibody to Bcl10. (B) HEK 293T cells were transfected with Bcl10, with (+) or without (−) Myr-Akt. Lysates were made, followed by IP of Bcl10. Immunoprecipitates were divided into two, and half were treated with lambda phosphatase (Ppase). SDS-PAGE and Bcl10 Western blotting were then performed. (C) Parental Jurkat T cells or CARMA1-deficient JPM 50.6 cells were transfected with Bcl10 and the indicated plasmids. Cell lysates were subjected to SDS-PAGE and Western blotting for Bcl10. The lower, open arrow indicates the location of nonphosphorylated Bcl10, while the top, closed arrows point to the phosphorylated forms of the protein. The positions of molecular mass markers (in kilodaltons) are indicated on the right-hand side of each blot. Results are representative of five (A), two (B), and three (C) experiments that were performed.
- FIG. 8.
Modulation of CARMA1-induced NF-κB by Akt. Jurkat T cells were transfected with an NF-κB-luciferase reporter and 7 μg of empty vector or CARMA1, plus either 5 μg of myristylated Akt (Myr-Akt) (A) or 3 μg of dominant-negative (DN) Akt (B). Luciferase activity was determined the next day, and is presented as the average number of light units from triplicate samples ± standard deviations (error bars) of a single experiment, representative of five experiments that were performed in each case. The inserts show Western blot analysis of Myc-CARMA1 expression. The blot in panel B includes an additional nonspecific band (lower band), because the anti-Myc (9E10) antibody used in this experiment was from a different source.
- FIG. 9.
Model for Akt participation in NF-κB induction by the TCR and CD28. Thick open arrows denote major pathways from which details have been omitted for simplicity. Curved lines indicate protein-protein interactions, which may or may not be direct, that have been demonstrated in this and other reports. Curved, dashed arrows indicate likely (not necessarily direct) phosphorylation events.
