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SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

Abstract

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1–RIAM–talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

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Figure 1: SHANK1 and SHANK3 inhibit β1-integrin activation.
Figure 2: Loss of SHANK1 and SHANK3 promotes cell adhesion and spreading.
Figure 3: Structural analysis of the SHANK3 SPN–ARR region.
Figure 4: SHANK3 interaction with Ras and Rap.
Figure 5: SHANK3 regulates integrin activation and cell spreading by a Rap1/Ras-dependent mechanism.
Figure 6: Loss of Shank3 promotes active Rap1 localization to the cell edge.
Figure 7: SHANK1 and SHANK3 sequester active Rap1 to inhibit RIAM–talin-dependent integrin activation and alter adhesion dynamics.
Figure 8: SHANK1 and SHANK3 regulate cell migration and invasion.

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Acknowledgements

We thank J. Jukkala, P. Laasola, J. Siivonen, H.-H. Hönck and V. Kolbe for technical assistance, M. Saari for help with the microscopes; C. Guzman for image and statistical analysis; G. Rosenberger (UKE Hamburg, Germany) for HA-tagged G-protein expression constructs; M. Matsuda (Kyoto University, Japan) for the Raichu-Rap1 probe; A. Shcheglovitov (Stanford University, California, USA) for pHAGE-EGFP-Shank3 construct; B. Baum (University College London, UK) and S. Royale (University of Warwick, UK) for R-Ras and Rap1a constructs; M. Davidson (University of California, USA) for mEmerald-paxillin construct and Cell Imaging Core facility, University of Turku Centre for Biotechnology for help with imaging. This study has been supported by the Academy of Finland (J.I., J.P., E.P.), an ERC Starting Grant, an ERC Consolidator Grant (615258), the Sigrid Juselius Foundation, the Finnish Cancer Organization (J.I.) and Deutsche Forschungsgemeinschaft (GRK1459; to H.-J.K.). J.L. is supported by the Turku Doctoral Programme of Molecular Medicine (TuDMM). T.Z. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) DTP fellowship. M.G. and G.J. are supported by an EMBO Long-Term Fellowship. F.H.N. is supported by DAAD; V.M. is supported by Dr. Hans Ritz und Lieselotte Ritz Stiftung; T.B. is supported by funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115300, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution (EU-AIMS) and by the BIU2 programme at Ulm University. We thank umif (Hamburg) for access to microscopes. The X-ray data were collected at the Diamond Light Source (UK) under Liverpool BAG allocation.

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Contributions

Conceptualization, J.I., H.-J.K. and I.L.B.; methodology, J.L., G.J., T.Z. and M.G.; formal analysis, T.Z., G.J., I.L.B. and J.L.; investigation, J.L., T.Z., M.G., G.J., N.D.F., E.P., M.B., V.M., F.H.N., J.I., H.-J.K. and I.L.B.; resources, T.B., J.I., H.-J.K. and I.L.B.; writing original draft, J.I., H.-J.K., I.L.B. and M.G.; writing–reviewing, J.I., J.L., M.G., G.J., H.-J.K., H.H. and I.L.B.; visualization, G.J., J.L., M.G., I.L.B., H.H. and H.-J.K.; supervision, J.P., J.I., H.-J.K. and I.L.B.; funding acquisition, J.I., H.-J.K. and I.L.B.

Corresponding authors

Correspondence to Hans-Juergen Kreienkamp, Igor L. Barsukov or Johanna Ivaska.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SHANK1 and SHANK3 inhibit β1-integrin activation.

a, Flow cytometric analysis of integrin activity in CHO cells upon transient expression of GFP or Shank1-GFP. Quantification reveals reduced integrin activation (FN7-10/total β1-integrin) upon transient expression of Shank1-GFP. Data represent mean ± s.e.m. expressed relative to GFP (n = 5 independent experiments; 5000 GFP-positive cells per experiment) b,c, SHANK3 and SHANK1 gene expression (mRNA levels) measured by Taqman qPCR and analysed using relative quantification (RQ) method in HEK293 and MDA-MD-321 cell lines. Data represent mean ± s.e.m. (n = 6 independent experiments). d, Representative immunoblots showing the efficiency of SHANK3 and SHANK1 silencing on protein expression in HEK293 and MDA-MB-231 cells, respectively. GAPDH was used as loading control. e, Flow cytometric analysis of integrin activity (9EG7/total β1-integrin) in MDA-MB-231 cells upon SHANK1 silencing and in the presence or absence of an exogenous integrin activator Mn2+. Data represent mean ± s.e.m. expressed relative to control-silenced cells (n = 9, 3, 9, 3 independent experiments from left to right; 10,000 cells per experiment) f, Flow cytometric analysis of integrin activity following re-expression of rat Shank1-GFP in SHANK1-silenced MDA-MB-231 cells. Quantification shows that re-expression of Shank1-GFP rescues the increase in integrin activity (9EG7/total β1-integrin) induced by SHANK1 silencing. Data represent mean ± s.e.m. expressed relative to control-silenced, GFP-expressing cells (n = 4 independent experiments; 5000 GFP-positive cells per experiment) g, Representative immunoblots of SHANK3 protein expression in isolated mouse mammary epithelial cells (MMEC) and fibroblasts (MMF) showing that SHANK3 is expressed in MMECs but not in MMFs. GAPDH was used as loading control. h, Flow cytometric analysis of integrin activity in MMECs. Representative histograms of show higher levels of active β1-integrin (9EG7, top panel) in MMECs isolated from Shank3αβ−/− (mutant) mice compared to Shank3αβ+/+ (WT) MMECs. No difference in total β1-integrin (MBI.2, bottom panel) was observed (data represent 1 out of 2 independent experiment; in each experiment cells were pooled from three mice). i, Flow cytometric analysis of integrin activity in MMFs. Representative histograms show equal levels of active (9EG7, top panel) and total (MBI.2, bottom panel) β1-integrin in MMFs isolated from Shank3αβ−/− (mutant) and Shank3αβ+/+ (WT) mice (data represent 1 of 2 independent experiment; in each experiment cells were pooled from three mice). Integrin activity is not significantly altered (mean of 2 independent experiment). j, Representative confocal images of MDA-MB-231 cells transiently expressing Shank3-mRFP or Shank1-GFP and adhering to fibronectin-collagen for 1 h. Confocal slices from the mid layer show that SHANK1 and SHANK3 localize with inactive integrin (MAB13) and F-actin in membrane ruffles. Scale bar: 10 μm. k, Representative confocal image of an MDA-MB-231 cell transiently expressing Shank3-mRFP plated on fibronectin-collagen for 1 h and stained for active β1-integrin (9EG7) and actin. Confocal slices from the bottom surface show that SHANK3 is excluded from active integrin-rich adhesions. Scale bar: 10 μm. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 2 Loss of SHANK1 and SHANK3 induces cell adhesion and spreading.

a, Rate of cell adherence (cell index) monitored in real-time using the xCELLigence system revealing that SHANK1 silencing in MDA-MB-231 cells promotes cell attachment on a fibronectin-collagen substrate. BSA was used as a control for background binding. Data represent mean ± s.e.m. (n = 4 independent experiments; average of four wells per experiment, 20,000 cells/well). b,c, Representative confocal images (b) and quantification (c) showing increased MDA-MB-231 cell spreading upon SHANK1silencing. Cells were plated on fibronectin-collagen, allowed to adhere and spread for 60 min, stained for F-actin and imaged using a TIRF microscope. Cell area was analysed using ImageJ software. Scale bar, 10 μm. Data are displayed as Tukey box plots of cell area relative to control-silenced cells (n = 116 cells from three independent experiments). d, Quantification of flow cytometry assay revealing reduced integrin activation (fibronectin7-10/total β1-integrin) upon transient expression of SHARPIN-GFP or Shank1-GFP in both WT and SHARPIN-null (cpdm) MEFs. Data are mean ± s.e.m. expressed relative to WT GFP expressing cells (n = 5 independent experiments; 5000 GFP-positive cells per experiment). Tukey box plots represent median and 25th and 75th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3.

Supplementary Figure 3 Folding assessment of SHANK3 SPN–ARR region.

a,b, Representative confocal images (a) and quantification (b) of active integrin levels in SK-N-BE-2 cells differentiated by 10 μM retinoid acid and plated on laminin for 3 days. Overexpression of Shank3 SPN WT, but not the SPNL68P mutant, reduces the intensity of active integrin (9EG7) staining in growth cones relative to area. Scale bar: 10 μm (a). Mean grey value (active integrin intensity) was analysed using the ImageJ software. Data represent mean ± s.e.m. and Tukey box plot are shown (n = 33, 27, 15 growth cones from left to right) (b). c, Sequence alignment of SHANK3 SPN and talin F0 domains showing sequence similarity in the UBL fold region. d, Superimposition of 1H, 15N-HSQC spectra comparing the SPN–ARR (red) region with isolated SPN (blue, upper panel) and ARR (blue, bottom panel) domains. These spectra show backbone HN-group signals that are highly sensitive to protein conformation and dynamics and are often called ‘fingerprint’ spectra. Folded proteins are characterized by highly dispersed signals of uniform intensity; with the increase in molecular weight, signals broaden out due to decreased protein mobility. The uniform resonance intensity of the SPN–ARR fragments indicated that the domains move as a single unit. In comparison, the signals of the isolated domains had smaller line-width and many of the signals did not overlap with any signals in the SPN–ARR fragment, while the signal distribution patterns were similar; in contrast, for a protein consisting of independently moving domains we would expect very similar positions and line-widths of signals of isolated domain and domain within a larger fragment. These spectral differences provide further support for the compact structure of the SPN–ARR unit, where the intramolecular contacts between SPN and ARR induce chemical shifts relative to the isolated domains and lead to reduced protein mobility. In this unit the SPN domain interactions are restricted to the protein surface exposed in the double domain structure. Tukey box plots represent median and 25th and 75th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test.

Supplementary Figure 4 Interaction of SHANK3 with Ras and Rap.

a, Western blot analysis of SHANK3 GFP-tagged SPN domain co-immunoprecipitation with the indicated GTPases (Rac and Cdc42 were T7-tagged and Rap1b was HA-tagged). Representative blots are shown. In: input, IP: immunoprecipitation. bf, ITC isotherms for active GTP-form H-Ras interaction with SHANK3 SPNWT-ARR (b) or SHANK3 SPN(R12C)-ARR mutant (c), inactive GDP-bound H-Ras interaction with SHANK3 SPNWT-ARR (d) or SHANK3 SPN(R12C)-ARR mutant (e) and active GTP-form of H-Ras interaction with SHANK1 SPNWT-ARR (f). Active GTP form of H-Ras was produced using non-hydrolysable GTP analogue GMPPCP. Solid lines indicate fitting to the single-site-binding model. g, Thermodynamic parameters for the different active G-protein/SHANK SPN binding pairs extracted from ITC binding curves. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 5 SHANK3 regulates integrin activation and cell spreading by a Rap1/Ras-dependent mechanism.

a, Flow cytometric analysis of integrin activity following transient co-expression of GFP-tagged constitutively active R-Ras mutant (R-RasG38V) together with WT or mutant Shank3-mRFP in HEK293 cells. Quantification shows that Shank3 WT-mRFP overexpression inhibits R-RasG38V-mediated increase in β1-integrin activity (9EG7/total β1-integrin). In contrast, expression of ASD-related Shank3 mutants (L68P, R12C) failed to oppose R-RasG38V-induced β1-integrin activation. Data are mean ± s.e.m. expressed relative to mRFP and GFP-expressing cells (n = 6, 6, 5, 5, 3 independent experiments from left to right; 5000 mRFP/GFP-positive cells analysed per experiment). b, Flow cytometric analysis of integrin activity shows that Mn2+-induced integrin activity is significantly impaired in Shank3 WT-mRFP overexpressing CHO cells. Data are mean ± s.e.m. expressed relative to mRFP-expressing cells (n = 4, 3, 4, 3 independent experiments from left to right; 5000 RFP-positive cells analysed per experiment). c, Total neurite length, main neurite length and number of end tips analysed from mouse cortex neurons plated on laminin. Data are mean ± s.e.m. (n = 7 independent experiments). Statistical analysis: Student’s t-test. Statistics source data can be found in Supplementary Table 3.

Supplementary Figure 6 SHANK3 silencing does not appear to affect total active Rap1 levels.

Lysates from control and SHANK3-silenced HEK293 cells were subjected to GST-RalGDS-RBD pulldown to isolate Rap1a/b-GTP (active Rap1). Total cell lysates were blotted for total Rap1a/b to normalize for Rap1 levels. Rap1-GTP levels are not significantly altered by SHANK3 silencing. Data are mean + / − s.e.m. (n = 3 independent experiments). Statistics source data can be found in Supplementary Table 3. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 SHANK1 and SHANK3 regulate cell migration and invasion.

ac, Random cell migration of MDA-MB-231 cells plated on a fibronectin-collagen matrix recorded over 24 h by time-lapse imaging showing that SHANK3-silenced cells migrate faster and more randomly than control-silenced cells. Representative cell tracks (a) and quantification of migration speed (b) and directionality (c) are shown. Data were analysed using the Manual Tracking plugin (ImageJ) and are displayed as Tukey box plots (n = 30 siCTRL and 24 siSHANK3 cells from 3 independent experiments). Tukey box plots represent median and 25th and 75th percentiles (interquartile range); points displayed as outliers if 1.5 times above or below the interquartile range; outliers are represented by dots. Statistical analysis: Student’s t-test.

Supplementary information

Supplementary Information

Supplementary Information (PDF 5759 kb)

Supplementary Table 1

Data collection and refinement statistics of the SHANK3 N-terminal domain. (PDF 97 kb)

Supplementary Table 2

List of antibodies and dilutions used in the study. (XLSX 12 kb)

Supplementary Table 3

Statistics source data. (XLSX 29 kb)

Analysis of Shank3 dynamics in cells.

MDA-MB-231 cells transiently expressing Shank3-mRFP were plated on fibronectin and imaged live using a spinning disk microscope (1 picture every 10 s). (M4V 6533 kb)

Shank regulation of cell adhesion dynamics: the talin ‘wave’.

Shank1-silenced or control-silenced MDA-MB-231 cells transiently expressing GFP-Talin-1 were plated on fibronectin and imaged live using a TIRF microscope (1 picture every 1 min for more than 3 h). (M4V 3388 kb)

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Lilja, J., Zacharchenko, T., Georgiadou, M. et al. SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras. Nat Cell Biol 19, 292–305 (2017). https://doi.org/10.1038/ncb3487

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