Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot–Marie–Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP–KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP–KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP–KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot–Marie–Tooth disease result from reduced CAP–KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.
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The mass-spectrometry proteomics data (Supplementary Tables 1, 2 and 3) have been deposited to the ProteomeXchange Consortium70 through the PRIDE partner repository under PX accession number PXD010484. Raw numerical data for the results in Figs. 4b, 5g–j and 6e are provided in Supplementary Table 4. Unprocessed blots and gels for all relevant figures are in Supplementary Fig. 7.
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We thank A. Hatch for conducting the initial pilot studies on BIF purification, Z. Svindrich and A. Lavanway for help with imaging and image analysis, T. Y. Chang for supplying neurons, W. Wickner for advice on protein purification, M. Pollak for input on FSGS, J. McLellan and M. Ragusa for comments and support, and C. Detteyala for the initial notion of actin post-translational modification. This work was supported by NIH R01 GM069818 and R35 GM122545 to H.N.H., R01 DK088826 to M. Pollak (HNH sub-contract), R35 GM119455 to A.N.K., and P20 GM113132 to the BioMT COBRE.
Integrated supplementary information
Supplementary Figure 1 Quantification of cytosolic actin polymerization by exogenously expressed INF2.
Confocal microscopy of U2OS cells transfected with GFP alone (vector), GFP-INF2-nonCAAX wildtype (WT) or A149D mutant. Fixed cells stained with TRITC-phalloidin (actin filaments). (a) Schematic of confocal imaging plane used. (b) Representative GFP and TRITC-phalloidin images. Arrows represent cytosolic regions to be quantified. Scale bar, 10 µm. (c) Normalized fluorescence intensity of TRITC-phalloidin. Lines represent mean (Vector: n = 31 cells, mean = 1.35, STD = 0.72; WT: n = 31 cells, mean = 1.19, STD = 1.02; A149D: n = 34 cells, mean = 2.48, STD = 2.14). p values determined by one-sided student’s t-test. N.S., no significant difference.
All panels conducted once, except panel a (First SourceQ step), which was conducted three times. (a) Graph showing protein elution profile (OD280 nm, grey) from first SourceQ step (pH 7.4) as well as the effect of each fraction on the activities of 20 nM INF2-FL (red) or FFC (green) in pyrene-actin assays. Activity of INF2-FL (blue), INF2-FFC (pink), or actin alone (black) are points on the left of graph. Fractions in purple box are those pooled for subsequent chromatography. Below graph are anti-CAP1 and CAP2 western blots of selected fractions. (b) Graph showing protein elution and activity profiles for the Superdex200 size exclusion step. Elution positions of standard proteins (thyroglobulin, ferritin, and γ-globulin) are indicated. Below graph are anti-CAP1 and CAP2 western blots of selected fractions. (c) Graph showing protein elution and activity profiles from the second SourceQ (pH 8.8). (d) Pyrene actin polymerization assay (2 µM actin monomer, 5% pyrene) testing effect of inhibitor (fraction #8 in fig. 2e) on 10 nM full-length INF2 or 10 nM mDia1 FFC. (e) Test of GFP-INF2 proteolysis in the presence of inhibitory fractions. Fractions were taken from pyrene-actin assays conducted in Fig. 2c, after completion of assay (1200 sec), and probed for INF2 by western blotting. (f) Pyrene actin polymerization assays (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL alone (green) or with an inhibitory fraction from final purification step (fraction #8 in Fig. 2e) that was either boiled (black) or not boiled (grey). Actin alone curve in red.
Supplementary Figure 3 Testing candidate proteins in the BIF for INF2 inhibition by immuno-depletion.
(a) Immuno-depletion of inhibitor candidates. An INF2 inhibitory fraction from mouse brain (Fraction #38–40 pool from SourceQ#1 (Supplementary Fig. 2a)) was incubated with either nonspecific antibody control (NS) or the following antibodies: (i) Vacuolar ATPase subunit A (VATA), (ii) Vacuolar ATPase subunit B2 (VATB2, partial depletion), or (iii) Heat shock cognate 71kDa protein (HSP7C). Experiment conducted once. (b) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL and supernatant fractions after immuno-depletion. IP/NS-Sup, NS depletion. IP/VATA-Sup, VATA depletion. IP/VATB2-Sup, VATB2 depletion. IP/HSP7C-Sup, HSP7C depletion. Pyrene-actin assays conducted once in duplicate.
(a) Coomassie stained SDS-PAGE of CAP1 or CAP2 purified from HEK293 cells, either as GFP-fusions or cleaved versions. Experiment conducted 6, 2, 24, and 20 times for CAP1–GFP, CAP1, CAP2–GFP, and CAP2, respectively. (b) Superdex200 gel filtration chromatography of purified CAP1 or CAP2 (cleaved from GFP). Elution positions of standard proteins (thyroglobulin, ferritin, and γ-globulin) indicated. Experiment conducted two times for CAP1 and 10 times for CAP2, respectively. (c) Velocity analytical ultracentrifugation of 7 µM CAP2–GFP. Mass: 726 kDa (calculated from sedimentation), 83.7 kDa (calculated from sequence). Experiment conducted once. (d) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL in the presence or absence of 5 µM indicated purified CAP proteins. Experiment conducted two times. (e) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) in the presence or absence of 5 µM indicated purified CAP proteins. Experiment conducted two times. (f) Pyrene actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM GFP-INF2-FFC in the presence or absence of 1 µM purified CAP either without actin exchange or exchanged with chicken muscle actin (CAP/CKSA). Experiment conducted three times.
(a) Schematic of exchange process, in which column-bound CAP/293A is mixed with TMR-actin. CAP/actin eluted by HRV3C digestion. (b) StrepTrap bead chromatographic columns containing no protein (left), CAP1–GFP (middle) and GFP alone (right). Top photo: before mixing beads with TMR-actin. Bottom photo: after mixing with TMR-actin and washing. Coomassie gel shows similar actin content in the HRV3C-eluted complexes after exchange. Experiment conducted twice. (c) Coomassie gel of purified chicken muscle actin and brain actin. Experiment conducted twice. (d) Pyrene-actin polymerization assay (2 µM actin) containing 20 nM GFP-INF2-FL and 3 µM profilin in the presence or absence of 1 µM purified CAP2/CSKA. Experiment conducted twice. (e) Coomassie-stained SDS/PAGE of HDAC6 purified from HEK293 cells. Experiment conducted three times. (f) Western blot probing tubulin and Ac-tubulin after treatment of purified brain tubulin (10 μM) with purified HDAC6 (200 nM) or buffer for 1 hr, 37 °C. Experiment conducted three times. (g) Western blot probing tubulin and Ac-tubulin in 293 lysate after culturing in medium containing DMSO or 50 μM Tubastatin A for 1 hr before lysis. Experiment conducted three times. (h) Western blot probing actin and Ac-lysine in recombinant CAP2–GFP purified from 293 cells incubated with either DMSO or 50 μM Tubastatin A before lysis. Experiment conducted three times. (i) vAUC of GFP-INF2-FFC (1.8 µM), with or w/o 18 µM CAP2/293-D or CAP2/293-T. Mass of GFP-INF2-FFC: 218.3 kDa (sedimentation), 112.6 kDa (sequence). Left axis: 490 nm (GFP-INF2), right axis: 280 nm (CAP). Experiment conducted once. (j) Representative GFP and TRITC-phalloidin confocal microscopy images showing cytosolic actin polymerization induced by exogenously expressed GFP-INF2 constructs (WT, L77R, or R218Q) in INF2-KO U2OS cells. Cells treated with either DMSO or 50 μM Tubastatin A for 1 hr followed by fixation and staining. Images are maximum intensity projections of three confocal imaging planes in the middle z region of the cell. Bar, 10 µm. (k) Normalized fluorescence intensity of TRITC-phalloidin quantified from images similar to (j). Lines represent mean. (WT-DMSO: n = 23 cells, mean = 1.34, STD = 0.64; WT-TubA: n = 20 cells, mean = 1.25, STD = 0.74; L77R-DMSO: n = 23 cells, mean = 6.76, STD = 2.33; L77R-TubA: n = 20 cells, mean = 7.06, STD = 2.38; R218Q-DMSO: n = 25 cells, mean = 13.35, STD = 4.60; R218Q-TubA: n = 29 cells, mean = 16.55, STD = 6.09). p-values determined by one-sided student’s t-test.
Actin in grey with pointed end up in all panels. K50, purple; K68, green; K215, orange; K315, blue. Approximate position of lysine 50 (not resolved in the structure) indicated by purple arrow. Models constructed using Pymol Anaconda2. (a) Actin monomer (PDB 4PGK). Actin sub-domains indicated by I, II, III and IV. Structure to right is 180° rotated from the left-hand structure. ATP shown as blue stick figure. (b) Actin filament (PDB 3G37). (c) Actin bound to CARP domain of mouse CAP1 (PDB 6FM2). Structure to right is 180˚ rotated from the left-hand structure. ADP shown as blue stick figure. (d) Actin bound to FH2 domain of mouse FMNL3 (PDB 4EAH).
Red boxes denote regions used in processed figure.
Supplementary Figures 1–7 and Supplementary Table titles and legends
Protein identification in the BIF fraction.
Acetylated lysine sites identified on chicken skeletal muscle actin bound to CAP2.
Comparison of acetylation on specific lysines between CAP and actin samples.
Raw data for large data sets.