PMC

Model for regulation of Cot by Akt. See the text for details.

Effects of mutated Cot constructs on kinase activity and transcriptional reporters. (A) Kinase activity of mutated kinase-inactive (KI) Cot constructs. 293T cells were transfected with 5 μg of the indicated myc-tagged Cot constructs. The next day anti-myc immunoprecipitates were washed and kinase activity was determined as described in Materials and Methods. Δ424 and Δ397 are the truncations referred to in the text. Equivalent levels of transfected Cot constructs were confirmed by Western blotting (data not shown). (B) Functional effects on inducible transcription of the Cot serine mutations were determined by cotransfection with RE/AP or AP-1 luciferase (Luc.) reporters in Jurkat T cells. WT, wild type.

Phosphorylation of Cot by Akt. (A) 293T cells were transfected with 5 μg of the indicated constructs. Glu epitope tag immunoprecipitates were incubated with kinase buffer and [³²P]ATP to allow phosphorylation of coprecipitated proteins. After SDS-PAGE, gels were transferred to PVDF and exposed to X-ray film (top gel), followed by rehydration and Western blotting with anti-myc antibody (bottom gel) to confirm the presence of coprecipitated kinase-inactive (KI) Cot proteins. (B) 293T cells were transfected with 5 μg of the indicated constructs, and the next day they were incubated with medium containing ³²P-orthophophoric acid. Transfected kinase-inactive Cot proteins were immunoprecipitated with anti-myc antibody, separated by SDS-PAGE, and transferred to PVDF. After exposure to X-ray film, membranes were probed with anti-myc antibody to confirm the presence of transfected Cot proteins. (C) The indicated constructs (5 μg) were transfected into Jurkat T cells with either the RE/AP or AP-1 luciferase reporter; luciferase activity was determined the next day as described in the text. FL, full length.

Mapping Akt-induced Cot phosphorylation sites. (A) Two-dimensional phosphopeptide analysis of Cot phosphorylated in vitro by Akt. The broken circle indicates the origin. A similar pattern of Cot phosphorylation was noted with samples phosphorylated in intact cells, although two additional spots appeared, consistent with the higher basal phosphorylation of Cot in intact cells (Fig. and data not shown). (B) Schematic diagram of the Cot domain structure and primary amino acid sequence. The two Akt-induced phosphorylation sites, determined as described in Materials and Methods, are underlined, and the sites of truncation described in the legend to Fig. are indicated by arrows. (C) Phosphorylation of Cot constructs by Akt. 293T cells were transfected with 5 μg of the indicated constructs, and in vitro phosphorylation was assessed as described for panel A. IP's, immunoprecipitates.

Functional interaction between Akt and Cot. (A) Jurkat T cells were transfected with the RE/AP luciferase (RE/AP-Luc.) reporter and 3 μg of wild-type (WT) Cot and/or 10 μg of wild-type Akt. The next day luciferase activity was determined as described in Materials and Methods. (B) Jurkat T cells were transfected with 10 μg of wild-type Akt, the indicated amounts of kinase-inactive (KI) Cot, and either RE/AP-luciferase (left panel) or pEF-GFP (right panel). The next day cells were stimulated with 10 ng of PMA/ml or were analyzed for GFP expression by flow cytometry. The inset in the left panel shows expression of Akt in the samples with RE/AP luciferase. (C) Hut78 cells were transfected with 10 μg of the indicated constructs and the RE/AP luciferase reporter. The inset shows constant levels of Cot expression in the relevant samples. (D) Jurkat cells stably transfected with a tetracycline-regulated PTEN allele were transfected with the RE/AP-luciferase reporter and 5 μg of the indicated constructs. Samples were split into wells without or with 1 μg of doxycycline (Dox)/ml. Twenty-four hours after transfection luciferase activity was determined. Myr. Akt, myristylated Akt.

Cot induction of p65/RelA nuclear entry. Jurkat (A) or HeLa (B) cells were transfected with the indicated Cot constructs and were stained with 9E10-Cy3 (to detect myc-tagged Cot) and Hoechst (to reveal nuclei) (images 1, 3, 5, and 7 in panel A and images 1 and 3 in panel B) and anti-p65 plus goat anti-rabbit-fluorescein isothiocyanate (images 2, 4, 6, and 8 in panel A and images 2 and 4 in panel B). (C) Quantitation of nuclear versus cytoplasmic localization of p65 in Cot-transfected HeLa cells. At least 40 Cot-transfected (red) cells of each type were scored for localization of p65 staining. Nuclear staining was scored when p65 intensity in the nucleus was greater than the overall staining intensity in the cytoplasm. Results in each part are representative of three independent experiments.

Specificity of NF-κB inhibition by Cot mutated at S400. (A) 293T cells were transfected with Flag-tagged IKKα and the indicated myc-tagged Cot or NIK constructs. Twenty hours after transfection cells were harvested, lysed, and subjected to immunoprecipitation with anti-Flag antibody M2. IKK kinase assays were conducted as described in Materials and Methods (upper gel). Aliquots of the lysates were assayed for expression of the myc-tagged proteins (lower gel). Equivalent levels of IKKα were confirmed by Western blotting (data not shown). (B and C) Dominant-negative activity of Cot SA(2). Jurkat T cells were transfected with an NF-κB-luciferase reporter and the indicated constructs. Cells were then stimulated for 6 h with 20 ng of PMA/ml (B), anti-CD3/CD28 (1 μg of anti-CD3/ml and 2 μg of anti-CD28/ml) (C), or 50 ng of TNF-α/ml (B and C), after which luciferase activity was determined. The results shown in panel C are shown as the percent maximal stimulation (versus vector control) and are the averages ± standard deviations of three experiments. WT, wild type; Autorad., autoradiography; WCL's, whole-cell lysates.

Physical interaction between Akt and Cot. 293T cells were transfected with 5 μg or the stated amount of the indicated plasmids. The next day cells were lysed and a small sample was removed to assess protein expression levels (lysates). The remainder was subjected to immunoprecipitation with Protein G beads covalently coupled to a monoclonal antibody against the Glu epitope tag (anti-Glu IP) of the transfected Akt construct (A) or the 9E10 monoclonal antibody against the myc epitope tag (anti-Myc IP) of the transfected Cot construct (B). Western blots of immunoprecipitated proteins and lysates were probed with anti-myc (A) or anti-HA antibody (B). (C) Tpl-2 constructs (2 to 10 μg; used in order to normalize expression levels) were analyzed for binding to Akt, as described for panel A. (D) Function of the Tpl-2 constructs was analyzed by cotransfection into Jurkat T cells with RE/AP or AP-1 luciferase (Luc.) reporters. Equal expression levels of the Tpl-2 constructs were confirmed by Western blotting (data not shown). IgH, immunoglobulin H; FL, full length; DC, deletion of C terminus.

Effects of Cot S400 mutation on IKK complex activation and association. (A and B) Activation of cotransfected (A) or endogenous (B) IKK by wild-type (WT) or SA(2) Cot. 293T cells were transfected with 5 μg of empty vector or the indicated Cot constructs; samples shown in panel A were also transfected with 5 μg of Flag-tagged IKKα or 0.1 μg of IKKβ. Twenty hours after transfection cells were harvested, lysed, and subjected to immunoprecipitation with either anti-Flag antibody M2 (A) or anti-IKKγ (B). IKK kinase assays were then carried out as described in Materials and Methods, with recombinant IκBα as an exogenous substrate. A small aliquot of each lysate was assayed by Western blot for expression of the Cot constructs (lower gels). Equal expression and immunoprecipitation of IKKα were confirmed by reprobing the kinase assay blot with anti-Flag antibody (data not shown). As IKKβ displays higher levels of kinase activity than IKKα, much lower quantities of the former were transfected, precluding detection of the autophosphorylated kinase (A, upper right gel). (C) 293T cells were cotransfected with 5 μg of Flag-tagged IKKα and the indicated Cot constructs. Twenty hours after transfection cells were harvested, lysed, and subjected to immunoprecipitation with anti-Flag antibody. After SDS-PAGE and Western blotting, membranes were probed with anti-Myc antibody 9E10 to reveal coprecipitated Cot. IP's, immunoprecipitates; FL, full length; Autorad., autoradiography; WCL, whole-cell lysates.