William C. Comb, Jessica E. Hutti, Patricia Cogswell, Lewis C. Cantley, and Albert S. Baldwin

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1 Molecular Cell, Volume 45 Supplemental Information p85 SH2 Domain Phosphorylation by IKK Promotes Feedback Inhibition of PI3K and Akt in Response to Cellular Starvation William C. Comb, Jessica E. Hutti, Patricia Cogswell, Lewis C. Cantley, and Albert S. Baldwin Inventory of Supplemental Information Figure 1 Figure S6 This figure demonstrates that Tak1 is not required for starvation-induced feedback of AKT. Figure 2 Figure S1 - In figure 2 we demonstrate that IKK is able to interact with and phosphorylate p85. This figure demonstrates that IKK can also interact with and phosphorylate p85 (S2 A and B respectively). Figure 3 Figure S2 This figure demonstrates that classic IKK stimuli TNF and LPS are also able to induce p85 Ser690 phosphorylation. Figure 4 Figure S3 This figure demonstrates IKK-dependent effects on AKT activity and is provided as a control for Figure 4A. Figure 5 Figure S4 (A) This figure demonstrates intrinsic ptyr affinity for WT and S690A csh2 domains and supports findings in Figure 5 that S690A has a decreased affinity for phospho-tyrosine compared to WT. (B) provides quantification for data presented in Figure 5D investigating p85-ptyr interactions in WT and IKK DKO cells in response to cellular starvation. Figure S5 This figure provides extra fluorescence images for WT p85 and S690A as presented in Figure 5. Statistical analyses for each experiment are also provided.

2 Figure S1, related to Figure 2 - IKK interacts with and phosphorylates p85a in vitro A. HEK293T cells were co-transfected with Flag-p85 and GST-IKK WT or kinasedead K44M as indicated. Flag immunoprecipitates were blotted with an anti-gst antibody. B. A bacterially purified GST-p85 C-terminal fragment (p50) containing the p85 csh2 was incubated with recombinant IKK (WT or K44M) as indicated. Kinase reactions were analyzed for incorporated 32P.

3 Figure S2, related to Figure 3 - Canonical IKK/NF- B stimuli TNF and LPS induce p85 Ser690 phosphorylation THP-1 monocytes were stimulated with TNF (10ng/mL) or LPS (200ng/mL) for the indicated timepoints. p85 immunoprecipitates were analyzed for phosphorylated Ser690. Whole cell extracts were also blotted with the indicated antibodies.

4 Figure S3, related to Figure 4 - Effects of IKK expression on Akt S473 phosphorylation Cos7 cells were transfected with Flag-p85 (WT or S690A), GST-IKK, and HA-Akt. Anti-HA immunoprecipitates were analyzed for Akt Ser473 phosphorylation.

5 Figure S4, related to Figure 5 - S690 and IKK requirements in p85 ptyr binding A. HEK293T cells were transfected with Flag-85 WT, S690A, or R649A. Cell lysates were incubated with a biotinyated phosphopeptide corresponding to P-Tyr472 of GAB1 or P-Tyr612 of IRS-1 (50nM). Peptide complexes were then precipitated with avidin conjugated sepharose beads. Coprecipitation of p85 WT or mutants with the P-Tyr peptides was evaluated by Flag immunoblot. B. Quantification of data in 5D, from 3 individual experiments. Densitometry was performed using imagej software on two bands labeled with arrowheads in Figure 5D. Data points were normalized to the percent of binding in untreated cells and the normalized values were averaged. Standard Deviation is represented.

6 Figure S5, related to Figure 5 - IKK expression is inversely correlated with PI3K membrane localization which reqires S690 A. EGFP-p85 WT and IKK were cotransfected into Cos7 cells. Represententative images are presented. Subcellular localization was monitored by flourescence microscopy. B. EGFP-p85 S690A and IKK were co-transfected into Cos7 cells. Represententative images are presented. Subcellular localization was monitored by flourescence microscopy. C. WT and S690A expressing cells from (A and B) were scored for p85 membrane localization and IKK expression levels. Weighted kappa statistical analyses were performed.

7 Figure S6, related to Figure 1 - Contribution of Tak1 to starvation-induced PI3K/Akt feedback WT and Tak1-deficient mefs were grown in starvation media for the indicated times and whole cell extracts were harvested for western blot analysis.