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1 Supplementary Materials for Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking Maeran Uhm, Merlijn Bazuine, Peng Zhao, Shian-Huey Chiang, Tingting Xiong, Sheelarani Karunanithi, Louise Chang, Alan R. Saltiel* *Corresponding author. Published 21 March 2017, Sci. Signal. 10, eaah5085 (2017) DOI: /scisignal.aah5085 The PDF file includes: Fig. S1. TBK1 activity is required for insulin-stimulated GLUT4 trafficking. Fig. S2. TBK1 phosphorylates Exo84 both in vitro and in cells. Fig. S3. TBK1 directly interacts with Exo84 through the coiled-coil domain of TBK1. Fig. S4. TBK1 does not affect RalA activity. Fig. S5. Mass spectrometry sequence coverage of affinity-purified phosphorylated HA-Exo84. Fig. S6. Temporal dynamics of the TBK1/exocyst/RalA complex in 3T3-L1 adipocytes in response to insulin. Other Supplementary Material for this manuscript includes the following: (available at Data files S1 and S2 (Microsoft Excel format). Identified phosphorylation sites of Exo84 from mass spectrometry.

2 Fig. S1. TBK1 activity is required for insulin-stimulated GLUT4 trafficking. (A) 3T3-L1 adipocytes pretreated with different doses of amlexanox were subjected to 2-DG uptake assay. Percent inhibition of insulin-stimulated glucose uptake was calculated and the values of IC 50 and maximal inhibition were produced with GraphPad Prism 6 software. Data are shown as the mean ± SEM. n = 3 experiments; data were analyzed by Mann-Whitney U test. (B) 3T3-L1 adipocytes pretreated with different doses of Cay were subjected to 2-DG uptake assay as shown in fig S1A. Data are shown as the mean ± SEM. n=3 biological replicates. (C) Cells were subjected to 2-DG uptake assay. Data are shown as the mean ± SEM. *p < 0.05; n = 3 experiments. (D) IB from fig S1C showing overexpression of WT TBK1 and its kinase-inactive mutant in 3T3-L1 adipocytes treated with or without 10 nm insulin for 30 minutes. n=3 experiments.

3 Fig. S2. TBK1 phosphorylates Exo84 both in vitro and in cells. (A) Schematic representation of Rattus norvegicus (Rat) Exo84. (B) Recombinant His-TBK1 was incubated with GST-Exo84 WT or myelin basic protein (MBP) for an in vitro kinase assay, resolved by SDS-PAGE, and detected by autoradiography. Lane 1: GST-Exo84 WT, Lane 2: His-TBK1 + GST-Exo84 with [ - 32 P]ATP, Lane 3: His-TBK1 + GST-Exo84 without [ - 32 P]ATP, Lane 4: MBP, Lane 5: His- TBK1 + MBP with [ - 32 P]ATP, and Lane 6: His-TBK1 + MBP without [ - 32 P]ATP. n=3 experiments. (C) GST-Exo84 RBD was cleaved with thrombin to generate GST-free Exo84 RBD. Lane 1: 1 g of GST-Exo84 RBD with thrombin, Lane 2: 500 ng of GST-Exo84 RBD with thrombin, Lane 3: 1 g of GST-Exo84 RBD, and Lane 4: GST. n=3 experiments. (D) Co-IP of MYC-TBK1 WT or the kinase-inactive mutant with HA-Exo84 from COS-1 cells. DE and LE stand for dark exposure and light exposure, respectively. n=3 experiments. (E) Cell lysates from COS-1 cells expressing Flag-TBK1 WT and the kinase-inactive mutant were incubated with immobilized GST, GST-RalBP1 RBD, GST-Exo84 RBD, or GST-Exo84 WT, subjected to GST pull-down assays, and analyzed by IB. Amount of GST fusion proteins used in the pull-down

4 assay was stained with Ponceau S. DE and LE stand for dark exposure and light exposure, respectively. n=3 experiments.

5 Fig. S3. TBK1 directly interacts with Exo84 through the coiled-coil domain of TBK1. (A) Co-IP of in vitro translated HA-tagged various truncation mutants of Exo84 with Flag-TBK1 WT or its kinase-inactive mutant. As a control, HA-empty vector was in vitro translated. Amount

6 of in vitro translated proteins used for IP is shown as input. Arrows indicate WT Exo84 and the Exo84 variants. Signals from IPed Exo84 Del 1 and Del 2 were masked by the heavy chain of the IgG as shown in the squares. DE and LE stand for dark exposure and light exposure, respectively. n=3 experiments. (B) Schematic representation of various domains in full-length and truncated TBK1. IB showing the amount of in vitro translated proteins used for Fig 3D and fig S3B. (C) Co-IP of in vitro translated MYC-tagged various truncation mutants of TBK1 with HA-WT Exo84 and its Del 5 mutant. As a control, MYC-empty vector was in vitro translated. Asterisks indicate WT TBK1 and arrow indicates Exo84 Del 5 mutant, respectively. n=3 experiments. (D) Co-IP of in vitro translated MYC-tagged various truncation mutants of TBK1 with HA-WT Exo84 and its Del 5 mutant. As a control, MYC-empty vector was in vitro translated. Asterisks indicate WT Exo84 and its Del 5 mutant co-iped with WT TBK1. The inactive kinase domain of TBK (KM) did not show phosphorylation of Ser 172 in TBK1 as shown in the squares. n=3 experiments.

7 Fig. S4. TBK1 does not affect RalA activity. Cell lysates from COS-1 cells co-expressing MYC-TBK1 WT and its kinase-inactive mutant with Flag-RalA WT and G23V (GV) mutant were incubated with GST-RalBP1 RBD beads, subjected to GST pull-down assays, and analyzed by IB. Amount of GST fusion proteins used in the pull down assay was stained with Ponceau S. DE and LE stand for dark exposure and light exposure, respectively. Normalized RalA activity (pull-down/total Flag-RalA, which was normalized to the third lane) is presented as the mean ± SEM. n = 3 experiments.

8 Fig. S5. Mass spectrometry sequence coverage of affinity-purified phosphorylated HA- Exo84. Identified sequence from mass spectrometry analysis is shown in gray. S or T indicates amino acid residue modified with phosphorylation. n=3 experiments.

9 Fig. S6. Temporal dynamics of the TBK1/exocyst/RalA complex in 3T3-L1 adipocytes in response to insulin. (A) Quantification of the binding of RalA with Exo84 as shown in Fig 8A, which was normalized to basal amounts at 0 minutes. *p < 0.05 as compared to basal amount; n

10 = 3 experiments. (B) Quantification of RalA activity as shown in Fig 8B. RalA activity (pulldown/total RalA) was normalized to the second lane. The normalized ratio is represented in arbitrary units. Data are shown as the mean ± SEM. *p < 0.05; n = 3 experiments. (C) Quantification of Exo84 phosphorylation as shown in Fig 8A. Phosphorylation was normalized to basal amounts at 0 minutes. *p < 0.05 as compared to basal amounts; n = 3 experiments. (D-F) Quantification of the binding of Sec8, Sec5, and TBK1 with Exo84 as shown in Fig 8A, which were normalized to basal amounts at 0 minute. *p < 0.05 as compared to basal amounts; n = 3 experiments; data were analyzed by Mann-Whitney U test.