Research ArticleCell Biology

MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity

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Science Signaling  31 Oct 2017:
Vol. 10, Issue 503, eaan3286
DOI: 10.1126/scisignal.aan3286
  • Fig. 1 Interaction networks of ARHGEF2 and MARK3.

    (A) ARHGEF2 high-confidence interactors detected by BioID MS (see table S1). Proteins with roles in similar biological processes are grouped by the indicated functions (see table S3), and reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. (B) MARK3 interactors detected by MS of immunoprecipitated Pyo-MARK3 complexes (see table S4). Reported protein-protein interactions (GeneMANIA) are highlighted with blue edges. (C) Pyo-tagged wild-type MARK3 and CNK1 (negative control) were coexpressed with CLASP1, CLASP2, and ARHGEF2 and immunoprecipitated (IP) from Cos cell lysates. The protein complexes were examined by Western blotting using specific antibodies for CLASP1, CLASP2, and ARHGEF2 and Pyo for MARK3. (D) Cell lysates from human embryonic kidney (HEK) 293T cells were immunoprecipitated using immunoglobulin G (IgG) or an antibody recognizing ARHGEF2 combined with Sepharose beads. The protein complexes were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and probed with antibodies recognizing MARK3 or ARHGEF2. Whole-cell lysates were analyzed by Western blotting to assess MARK3 and ARHGEF2 protein abundance. (E) KSR1 N′ (N-terminal head domain of KSR1), CLASP1, CLASP2, and ARHGEF2 proteins were immunoprecipitated from Cos cells and incubated with purified active wild-type (WT) or kinase-dead (KD) MARK3 in the presence of [γ-32P]ATP. The labeled proteins were visualized by autoradiography and also Western-blotted to detect the purified MARK3 proteins. (C to E) Data are representative of three independent experiments. (F) Analysis of the protein sequences of CLASP1, CLASP2, and ARHGEF2 for the consensus MARK3 phosphorylation motifs ΦaxRxxS*ΦPxxΦa and ΦaxR/KxxS*xxxΦa using ScanProsite (S* is the site phosphorylated, x is any amino acid, Φa is a hydrophobic residue with an aliphatic side chain, and Φ is any hydrophobic amino acid).

  • Fig. 2 MARK3 binds an N-terminal region of ARHGEF2 and phosphorylates Ser151.

    (A) Left: Flag-tagged ARHGEF2 fragments and wild-type MARK3 were coexpressed in HEK293T cells. Protein complexes were immunoprecipitated, and immunoblots were probed with antibodies specific for MARK3 and pan 14-3-3 to map the interaction. Antibodies recognizing Flag, MARK, and 14-3-3 antibodies were used to detect protein abundance in cell lysates, and α-tubulin was used as a loading control. Right: Schematic representation of the constructs used for mapping the interaction. EV, empty vector; WCL, whole-cell lysates; F.L., full length. (B and C) ARHGEF2 wild-type (top) or mutant ARHGEF2S151A (bottom) proteins were incubated with [32P]ATP, digested with trypsin, and examined by high-performance liquid chromatography (HPLC) analysis. For the in vitro analysis (B), purified MARK3 was added. CPM, counts per minute. (D) Purified ARHGEF2 mutants were incubated with purified active MARK3 in the presence of [32P]ATP. For each mutant, the [32P]phosphate incorporated was quantitated using a phosphoimager. (A to D) Data are representative of three independent experiments. (E) Alignment of ARHGEF2 orthologs in vertebrates. The asterisks represent sequences reviewed by Swiss-Prot. The boxed area shows the consensus motif, and the color code is based on the conserved residues, with red being the most conserved. The full alignment is shown in fig. S2.

  • Fig. 3 MARK3 perturbs the interaction between DYNLT1 and ARHGEF2 and stimulates exchange activity.

    (A) MST binding assays of phosphorylated and unphosphorylated FITC-labeled ARHGEF2 peptides for Ser885 (amino acids 876 to 891; top) and Ser151 (amino acids 142 to 157; bottom). The peptides were prepared at 100 nM with increasing concentrations of glutathione S-transferase (GST)–14-3-3. Kd values were determined from the thermophoresis titration curves for the phosphorylated peptides, whereas no binding was detected for unphosphorylated peptides. Kd values are the average of three independent experiments ± SD. (B) Green fluorescent protein (GFP)–tagged wild-type ARHGEF2 and a truncated version (deletion of residues 87 to 151) were coexpressed with Pyo-tagged wild-type MARK3 in HEK293T cells. Protein complexes were immunoprecipitated using an antibody specific for GFP, and immunoblots were probed with antibodies recognizing Pyo to detect interactions with MARK3. Antibodies specific for GFP and Pyo were used to detect protein abundance in whole-cell lysates. Bottom: Quantification of the interaction normalized with total lysate. Data are means ± SD of three independent experiments. (C) Myc-tagged ARHGEF2 was coexpressed with Flag-tagged DYNLT1 in the absence or presence of increasing amounts of wild-type or kinase-deficient MARK3. Protein complexes were immunoprecipitated using an antibody recognizing Myc antibody and analyzed by Western blotting for the presence of MARK3, DYNLT1, and endogenous 14-3-3 using antibodies against MARK3, Flag, and pan 14-3-3, respectively. Whole-cell lysates were analyzed by Western blotting and probed with the same antibodies to assess protein abundance, and α-tubulin was used as a loading control (see also fig. S3, A and B, for quantification). Data are representative of four independent experiments. (D) Myc-tagged wild-type ARHGEF2 and S151A and S885A mutants were coexpressed with Flag-tagged DYNLT1 in the absence or presence of increasing amounts of wild-type MARK3 in HEK293T cells. Protein complexes were immunoprecipitated using an antibody recognizing Myc and analyzed by Western blotting for the presence of MARK3, DYNLT1, and endogenous 14-3-3 using antibodies against MARK3, Flag, and 14-3-3, respectively. Whole-cell lysates were analyzed with the same antibodies to assess protein abundance. Total extracellular signal–regulated kinase (ERK) was used as a loading control (see fig. S3C for quantification). Data are representative of three independent experiments. (E) Myc-tagged wild-type and phosphomimetic mutants S151D and S151E for ARHGEF2 were coexpressed with Flag-tagged DYNLT1. Protein complexes were immunoprecipitated with an antibody recognizing Myc and analyzed by Western blotting. Antibodies against Myc, Flag, and 14-3-3 were used to confirm the amount of ARHGEF2 and to detect DYNLT1 and endogenous 14-3-3 in the complexes, respectively. Protein abundance in whole-cell lysates was analyzed using the same antibodies, and α-tubulin was used as a loading control. Data are representative of three independent experiments. (F) NMR-based GEF assays were performed to measure RHOA exchange rates in the presence of cell lysates from HEK293T cells expressing GFP alone; GFP-ARHGEF2; GFP-ARHGEF2 and Pyo-MARK3; or GFP-ARHGEF2, Pyo-MARK3, and Flag-14-3-3. The amount of ARHGEF2 in exchange assays was normalized on the basis of GFP fluorescence in the lysate, and protein amounts were detected by Western blotting (inset). The rates were normalized to ARHGEF2 exchange rate. Data are means ± SD of five independent experiments. Statistical significance was determined by a Kruskal-Wallis test with a Dunn’s posttest correction for multiple comparisons. *P = 0.0151 (ARHGEF2 compared to ARHGEF2 + MARK3); **P = 0.0072 (ARHGEF2 compared to ARHGEF2 + MARK3 + 14-3-3); **P = 0.0079 (GFP compared to ARHGEF2). NS, not significant. (G) Nucleotide exchange rates for RHOA in the presence of cell lysates from HEK293T cells expressing GFP-ARHGEF2WT or GFP-tagged ARHGEF2S151A. The rates were normalized to ARHGEF2WT exchange rate. Data are means ± SD of four independent experiments. Statistical significance was determined by a Mann-Whitney test. *P = 0.0286.

  • Fig. 4 Structural characterization of the DYNLT1-ARHGEF2 interaction.

    (A) Fluorescence polarization binding assays were performed with FITC-labeled ARHGEF2 peptides (residues 142 to 157) with and without the phosphorylation of Ser151. The peptides were titrated with increasing amounts of recombinant GST-DYNLT1. Data are representative of two independent experiments. mP, millipolarization units. (B) Overlay of 1H-15N HSQC spectra of DYNLT1 in the absence (blue) or presence (red) of mARHGEF2 peptide (residues 136 to 164) or in the context of a DYNLT1:mARHGEF2 chimera with a single glycine linker (black). Inset boxes (a and b) zoom into the overlay of the spectra. Chemical shift changes for selected residues are highlighted with arrows in the spectra. ppm, parts per million. (C) Structure of DYNLT1:ARHGEF2 chimera [Protein Data Bank (PDB): 5WI4] (see fig. S4C for the asymmetric unit and crystallographic contact sites). Ribbon representation: Blue and cyan are used to distinguish the two subunits of DYNLT1. The ARHGEF2 portion is yellow. (D) Schematic representation of interactions between the domain-swapped β strand of DYNLT1 and ARHGEF2. Residues of DYNLT1 (gray) and ARHGEF2 (yellow) with the hydrogen bond network are shown by black dashed lines. A kink in ARHGEF2 is caused by the insertion (relative to a perfect β strand) of residues Val150 and Ser151 (cyan box), which form a β bulge. (E) Detail of DYNLT1 in complex with ARHGEF2. Enlargement showing the ribbon representation of DYNLT1 (blue) with a stick model of the mARHGEF2 (yellow) component of the chimera (PDB: 5WI4). ARHGEF2 residues Leu146 to Asn154 are highlighted.

  • Fig. 5 The LKB1-MARK3 axis and PP2A regulate the phosphorylation of ARHGEF2 Ser151.

    (A) Western blot of HEK293T cells treated with dimethyl sulfoxide (DMSO), the PP2A inhibitor OA (50 nM for 4 hours), and the AMPK activator AICAR (1 mM for 6 hours). Phosphorylated ARHGEF2 Ser151 was detected using a site-specific antibody (see fig. S5A for antibody characterization), and total ERK was used as a loading control. Bottom: Quantification of the phosphorylation normalized to total ARHGEF2. Data are means ± SD of three independent experiments. (B) Western blot of HEK293T cells overexpressing Pyo-tagged MARK3WT or Flag-tagged PPP2R5B. Phosphorylated ARHGEF2 Ser151 and Ser885 were detected using site-specific antibodies, and α-tubulin was used as a loading control. Bottom: Quantification of Ser151 and Ser885 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments. (C) Western blot of 293 Flp-In T-REx cell lines carrying inducible expression of Flag-tagged GFP, the PP2A catalytic subunit PPP2CB (CB), or the regulatory B′ subunit PPP2R5B (5B). The cells were induced overnight with tetracycline (500 ng/ml) and transfected with empty vector, pyo-MARK3WT, or pyo-MARK3KD. Phosphorylated ARHGEF2 Ser151 was detected using a site-specific antibody, and α-tubulin was used as a loading control. Bottom: Quantification of the phosphorylation normalized to total ARHGEF2. Data are means ± SD of four independent experiments. (D) Western blot of A549 LKB1-deficient cells stably expressing empty vector (pBabe), wild-type LKB1 (LKB1WT), or kinase-deficient LKB1 (LKB1KD). Phosphorylation of AMPK was used as a control substrate for LKB1 phosphorylation. Phosphorylated ARHGEF2 Ser151 and AMPK Thr172 were detected using site-specific antibodies, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Bottom: Quantification of ARHGEF2 Ser151 phosphorylation normalized with total ARHGEF2. PBP, pBabe-puromycin (empty vector). Data are means ± SD of three independent experiments. (E) Western blot of A549 cells expressing LKB1WT and treated with a small interfering RNA (siRNA) pool specific for MARK3 or control siRNA for 72 hours. AMPK was used as a control substrate for LKB1-mediated phosphorylation. GAPDH was used as a loading control. Bottom: Quantification of ARHGEF2 Ser151 phosphorylation normalized with total ARHGEF2. Data are means ± SD of three independent experiments.

  • Fig. 6 MARK3 affects the localization of ARHGEF2 dependent on Ser151 and 14-3-3.

    (A and B) Live imaging of HEK293T cells. (A) Top: Cells transiently overexpressing GFP alone and GFP-ARHGEF2WT, alone or in combination with Cherry-MARK3; bottom: cells expressing GFP-ARHGEF2S151A alone or coexpressed with Cherry-MARK3. ARHGEF2 distribution in (A) is quantified in (B) (as percentage of cells). A total of 100 to 200 cells per condition were counted. Data are means ± SD of three independent experiments (n = 3). Scale bars, 10 μm. Statistical significance was determined by a two-way analysis of variance (ANOVA) test with a Bonferroni posttest correction for multiple comparisons. ****P ≤ 0.0001. (C and D) Live imaging of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2WT, pLVX-GFP ARHGEF2S151A, and pLVX-GFP ARHGEF2WT in combination with siMARK3; zoomed regions are shown in bottom panels. Scale bars, 20 μm (top) and 10 μm (bottom). (D) Quantification, as percentage of cells, of images in (C): cells having a higher tendency of showing a filament-like distribution (F > D), a higher tendency of having a diffuse distribution (D > F), or similar distribution of filament-like and diffuse-appearing structures (D = F). F, filament-like distribution; D, diffuse distribution. A total of 250 to 300 cells per condition were counted. Data are means ± SD of three independent experiments. Statistical significance was determined by a two-way ANOVA test with a Bonferroni posttest correction for multiple comparisons. *P = 0.0470 (in F > D; ARHGEF2WT compared to ARHGEF2WT + siMARK3); *P = 0.0235 (in F = D; ARHGEF2WT compared to ARHGEF2WT + siMARK3); **P = 0.0014; ***P = 0.0002 (in F > D; ARHGEF2WT + siMARK3 versus ARHGEF2S151A); ***P = 0.0005 (in D > F; ARHGEF2WT compared to ARHGEF2WT + siMARK3); ****P ≤ 0.0001.

  • Fig. 7 MARK3 phosphorylation of ARHGEF2 Ser151 regulates several biological functions.

    (A and B) Immunofluorescence of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2WT, pLVX-GFP ARHGEF2S151A, and pLVX-GFP ARHGEF2WT with siMARK3. Left: The cells were fixed and stained for actin; GFP signal is shown (inset). Right: Lookup tables showing the fluorescence intensity, with white denoting maximum intensity. (B) Quantification of the mean fluorescence intensity of five high-magnification fields per condition and per experiment for the images in (A). Data are means ± SD of three independent experiments. Scale bar, 20 μm. Statistical significance was determined by a one-way ANOVA test with a Bonferroni posttest correction for multiple comparisons. *P = 0.0453; ****P ≤ 0.0001. (C and D) Immunofluorescence of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2WT, pLVX-GFP ARHGEF2S151A, and pLVX-GFP ARHGEF2WT with siMARK3. The cells were fixed and stained for vinculin; GFP signal is shown (inset). (D) Quantification of the average size of the focal adhesion (FA) from four high-magnification fields per condition and per experiment of the images shown in (C). Data are means ± SD of three independent experiments. Scale bar, 20 μm. Statistical significance was determined by a one-way ANOVA test with a Bonferroni posttest correction for multiple comparisons. ***P = 0.0010; ****P ≤ 0.0001. (E) Relative wound density (cell density in the wound area expressed relative to the cell density outside of the wound area over time), in percentage, of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2WT, and pLVX-GFP ARHGEF2S151A exposed to increasing amounts of doxycycline (Dox) at 6 and 18 hours. Bottom: Comparison of the wounds at 18 hours. Blue, mask of the original wound; orange, wound not closed. Data are means ± SD of three independent experiments done in triplicates. Statistical significance was determined by a two-way ANOVA test with a Bonferroni posttest correction for multiple comparisons. *P = 0.0445; **P = 0.0029; ****P ≤ 0.0001. NT, no treatment.

  • Fig. 8 Phosphorylation of ARHGEF2 Ser151 is required for normal cell polarity.

    (A to C) 3D culture of MDCKII cells stably expressing inducible pLVX-GFP, pLVX-GFP ARHGEF2WT, and pLVX-GFP ARHGEF2S151A. (A) GFP fluorescence was visualized and cysts were stained for E-cadherin, actin, and 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 μm. (B) Average size of the cysts observed in pLVX-GFP, pLVX-GFP ARHGEF2WT, and pLVX-GFP ARHGEF2S151A (n = 24, 21, and 24, respectively). Data are means ± SD of three independent experiments. Statistical significance was determined by a one-way ANOVA test with a Bonferroni posttest correction for multiple comparisons. ****P ≤ 0.0001. (C) Z-stacks (1 μm) of pLVX-GFP ARHGEF2WT cysts. Abnormal mitotic events are indicated (yellow arrows). Note that the cyst on the left has lost expression of pLVX-GFP ARHGEF2WT. Numbers represent the Z-stack step. Images are representative of four independent experiments. Scale bar, 20 μm. (D) Model of MARK3- and PP2A-mediated regulation of ARHGEF2 phosphorylation and its effects on RHOA activation. LKB1 activates MARK3, which in turn phosphorylates ARHGEF2 on Ser151. This creates a 14-3-3 binding site that disrupts ARHGEF2 interaction with DYNLT1 and releases it from microtubules to activate RHOA and trigger the formation of stress fibers and focal adhesions. MARK3-mediated phosphorylation of Ser151 is required for epithelial cell polarity in 3D growth. PP2A dephosphorylates Ser151 through interactions with the B′ subunits. GDP, guanosine diphosphate; GTP, guanosine triphosphate.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/503/eaan3286/DC1

    Fig. S1. ARHGEF2 interaction network.

    Fig. S2. Full alignment of ARHGEF2 orthologs in vertebrates.

    Fig. S3. Quantitative analysis of the effect of MARK3 phosphorylation of ARHGEF2 on its interactions with DYNLT1 and 14-3-3.

    Fig. S4. Structural characterization of the DYNLT1-ARHGEF2 interaction.

    Fig. S5. MARK3, but not PP6, regulates the phosphorylation of ARHGEF2 Ser151.

    Fig. S6. Generation and validation of inducible cells.

    Fig. S7. MARK3 affects the biological activity of ARHGEF2.

    Fig. S8. Phosphorylation of ARHGEF2 Ser151 is required for normal cell polarity.

    Table S1. Summary of ARHGEF2 interactors reported in this study.

    Table S2. GO enrichment analysis of the ARHGEF2 network.

    Table S3. Functional annotation of ARHGEF2 interactors.

    Table S4. Analysis of MARK3–C-TAK1 complexes.

    Table S5. Data collection and refinement statistics for the DYNLT1:ARHGEF2 chimera (PDB: 5WI4).

    Data file S1. Raw data for the ARHGEF2 interactors reported in this study.

  • Supplementary Materials for:

    MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity

    María-José Sandí, Christopher B. Marshall, Marc Balan, Étienne Coyaud, Ming Zhou, Daniel M. Monson, Noboru Ishiyama, Arun A. Chandrakumar, José La Rose, Amber L. Couzens, Anne-Claude Gingras, Brian Raught, Wei Xu, Mitsuhiko Ikura, Deborah K. Morrison, Robert Rottapel*

    *Corresponding author. Email: rottapel{at}uhnresearch.ca

    This PDF file includes:

    • Fig. S1. ARHGEF2 interaction network.
    • Fig. S2. Full alignment of ARHGEF2 orthologs in vertebrates.
    • Fig. S3. Quantitative analysis of the effect of MARK3 phosphorylation of ARHGEF2 on its interactions with DYNLT1 and 14-3-3.
    • Fig. S4. Structural characterization of the DYNLT1-ARHGEF2 interaction.
    • Fig. S5. MARK3, but not PP6, regulates the phosphorylation of ARHGEF2 Ser151.
    • Fig. S6. Generation and validation of inducible cells.
    • Fig. S7. MARK3 affects the biological activity of ARHGEF2.
    • Fig. S8. Phosphorylation of ARHGEF2 Ser151 is required for normal cell polarity.
    • Table S1. Summary of ARHGEF2 interactors reported in this study.
    • Table S2. GO enrichment analysis of the ARHGEF2 network.
    • Table S3. Functional annotation of ARHGEF2 interactors.
    • Table S4. Analysis of MARK3–C-TAK1 complexes.
    • Table S5. Data collection and refinement statistics for the DYNLT1:ARHGEF2 chimera (PDB: 5WI4).

    [Download PDF]

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    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Raw data for the ARHGEF2 interactors reported in this study.

    Citation: M.-J. Sandí, C. B. Marshall, M. Balan, É. Coyaud, M. Zhou, D. M. Monson, N. Ishiyama, A. A. Chandrakumar, J. La Rose, A. L. Couzens, A.-C. Gingras, B. Raught, W. Xu, M. Ikura, D. K. Morrison, R. Rottapel, MARK3-mediated phosphorylation of ARHGEF2 couples microtubules to the actin cytoskeleton to establish cell polarity. Sci. Signal. 10, eaan3286 (2017).

    © 2017 American Association for the Advancement of Science

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