Journal: Journal of Biomedical Science

Article Title: Helicobacter pylori -induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation

doi: 10.1186/s12929-021-00710-0

Figure Lengend Snippet: MRCKβ is decreased in H. pylori- mediated GC and interacts with Siah2. a Western blot of whole cell extracts from uninfected and H. pylori -infected MKN45 cells (at 6, 12, and 24 h of infection with 200 MOI of H. pylori 26695) showing protein level of MRCKβ. GAPDH is a loading control. Bar graph clearly indicates a time-dependent decrease of MRCKβ after H. pylori infection. Two-way ANOVA followed by Tukey’s post hoc analysis is used to determine statistical significance. b Western blotting of total cell lysate from uninfected and H. pylori- infected (at MOI 100, 200 and 300) MKN45 cells showing MRCKβ protein. GAPDH is used as a loading control. Graph represents decrease of MRCKβ protein as compared to uninfected control. One-way ANOVA is used to determine statistical significance. c A representative western blot of infected (200 MOI for 12 h of H. pylori 26695 or 8–1 strain) or uninfected MKN45 cells showing MRCKβ protein level (n = 3). GAPDH is used as a loading control. d A representative (n = 9) immunofluorescence micrograph of human metastatic GC biopsy tissue samples showing the status of MRCKβ. Tissues are sectioned at 5 μm thickness. Images are captured using 40X objective and scale bars = 50 μm. e Western blot of whole cell lysate from uninfected and infected ( H. pylori at 200 MOI for 12 h) MKN45 cells subjected to co-immunoprecipitation using Siah2 antibody reveals that Siah2 interacts with MRCKβ. IgG band is used to indicate equal loading. f A representative western analysis indicating ubiquitination status of MRCKβ from uninfected or infected ( H. pylori at 200 MOI for 12 h) and 50 µM MG132-treated MKN45 cells. Reprobed bands of MRCKβ, Siah2 and GAPDH. g Western blotting of total cell lysates from uninfected or infected ( H. pylori at 200 MOI for 12 h) and 50 µM MG132-treated MKN45 cells subjected to co-immunoprecipitation using Siah2 antibody showing MRCKβ and Siah2 protein status. IgG band is used as the indicator of equal loading. All data are mean ± sem (n = 3). ** P < 0.01, *** P < 0.001

Article Snippet: When required, cells were transfected with control siRNA (Santa Cruz Biotechnology, TX) and mrckβ siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000.

Techniques: Western Blot, Infection, Control, Immunofluorescence, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Biomedical Science

Article Title: Helicobacter pylori -induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation

doi: 10.1186/s12929-021-00710-0

Figure Lengend Snippet: MRCKβ is degraded by Siah2. a Representative (n = 3) fluorescence microscopy of pcDNA3.1+ and siah2 WT stably-expressing MKN45 cells infected with H. pylori (200 MOI for 12 h). Images are captured using 60X objective and scale bars represent 20 μm. Bar graphs represent mean fluorescence intensity of Siah2 and MRCKβ obtained from cells of three independent experiments. Two-way ANOVA is used to determine statistical significance. All data are mean ± sem (n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001. b A representative western blot of control siRNA and siah2 siRNA-transfected MKN45 cells followed by 12 h H. pylori infection showing MRCKβ and Siah2 protein levels. GAPDH is used as a loading control

Article Snippet: When required, cells were transfected with control siRNA (Santa Cruz Biotechnology, TX) and mrckβ siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000.

Techniques: Fluorescence, Microscopy, Stable Transfection, Expressing, Infection, Western Blot, Control, Transfection

Journal: Journal of Biomedical Science

Article Title: Helicobacter pylori -induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation

doi: 10.1186/s12929-021-00710-0

Figure Lengend Snippet: MRCKβ potentiates Siah2 phosphorylation in GECs after H. pylori infection. a Confocal images (n = 3) of uninfected and H. pylori -challenged (200 MOI for 12 h) AGS cells representing subcellular localization of Siah2 and MRCKβ proteins. Individual DAPI-stained panels showing nuclear staining are not shown due to constraint of space but the merged panels include DAPI. Scale bar represents 5 μm. Scatter plots generated by using NIS AR software represent co-localization of MRCKβ and Siah2 along with their Pearson's correlation (R) and Mander’s overlap (MO) values. b A representative (n = 3) western blot of whole cell lysates of MKN45 cells transfected with pcDNA3.1+ and mrckβ and infected with H. pylori showing MRCKβ, P-Ser-Siah2, P-Thr-Siah2 and Siah2 protein status. GAPDH = loading control. Bar graphs indicate increase of P-Ser-Siah2, P-Thr-Siah2 and Siah2 after mrckβ overexpression followed by 12 h of H. pylori infection. Two-way ANOVA followed by Tukey’s post hoc analysis are performed to evaluate statistical significance. Data are mean ± sem (n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001. c Representative western blot (n = 3) of pcDNA3.1+, siah2 WT and Siah2 phospho-null mutant-expressing MKN45 stable cells transfected with mrckβ followed by H. pylori infection showing P-Ser-Siah2 and Siah2 (above) and P-Thr-Siah2 and Siah2 (below). GAPDH is the loading control. d Representative western blot (n = 3) from whole cell lysate of MKN45 cells transfected with either control or mrckβ siRNA followed by H. pylori infection indicating MRCKβ, P-Ser 6 -Siah2, P-Thr 279 -Siah2 and Siah2 proteins. GAPDH is kept as a loading control

Article Snippet: When required, cells were transfected with control siRNA (Santa Cruz Biotechnology, TX) and mrckβ siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000.

Techniques: Phospho-proteomics, Infection, Staining, Generated, Software, Western Blot, Transfection, Control, Over Expression, Mutagenesis, Expressing

Journal: Journal of Biomedical Science

Article Title: Helicobacter pylori -induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation

doi: 10.1186/s12929-021-00710-0

Figure Lengend Snippet: H. felis -infected C57BL/6 mice exhibit enhanced Siah2 phosphorylation and MRCKβ downregulation. a Immunofluorescence microscopy images of antral gastric tissues of uninfected and infected mice showing the status of Siah2 and MRCKβ. b Immunofluorescence microscopy images of antral gastric tissues of C57BL/6 mice showing the status of MRCKβ, P-Ser 6 -Siah2 and P-Thr 279 -Siah2. All tissues are sectioned at 5 μm thickness. All images are captured using 10× objective and scale bars represent 100 μm

Article Snippet: When required, cells were transfected with control siRNA (Santa Cruz Biotechnology, TX) and mrckβ siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000.

Techniques: Infection, Phospho-proteomics, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes treated for 1 h with DMSO, ML-141 (5 μM), BDP-9066 (1 μM) or a combination of BDP-9066 (1 μM) and calyculin A (CalA; 0.5 nM). Images are single confocal frames taken across metaphase chromosomes. (B) Immunofluorescence detection of endogenous MRCKbeta in MII oocytes. The top row shows a control oocyte treated with DMSO. An enlarged view of the polarized cortex is shown on the right. The bottom row shows an oocyte treated with ML-141 (5 μM). (C) Bar graph depicting the fraction of oocytes showing polarized accumulation of MRCKbeta, in MII oocytes treated with DMSO, ML-141 or BDP-9066. P values were calculated using Fisher’s exact test. (D) Immunofluorescence detection of MRCKα in a mouse MII oocyte. The dotted line denotes the oocyte perimeter. No cortical staining could be detected. The image is representative of 33 similar observations. (E) Localization of MRCKβ-EGFP (left) and MRCKβ H1593/1596A-EGFP (right) in MII oocytes. Note the absence of cortical localization for the Cdc42 binding-deficient MRCKβ. Images are representative of over 40 similar observations. (F) Immunofluorescence detection of activated myosin II (P-MRLC) in MII oocytes expressing MRCKβ-K105A. A majority of oocytes showed a complete loss of the P-MRLC ring (62.5%; top row), while the remaining oocytes showed incomplete inhibition (37.5%; bottom row, white arrow). (G) Bar graph depicting the percentage of MII oocytes showing a P-MRLC ring, in various experimental conditions as shown in (A), (E) and (F). P values were calculated using Fisher’s exact test. (H) Immunofluorescence detection of tubulin showing spindle localization in MII oocytes treated with DMSO (left), CK-666 (100 μM) for 3h (middle), and CK-666 (100 μM) and BDP-9066 (1 μM) for 3h (right). The yellow line shows the distance (d) separating the maternal chromosomes from the nearest cortex. (I) Box plot showing the distance between maternal chromosomes and the nearest cortical region in oocytes treated with DMSO (controls), CK-666 alone, CK-666 and BDP-9066, and in oocytes expressing MRCKβ-K105A treated with CK-666. P values were calculated using Student’s t -test. NS: non significant. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3. The number of oocytes scored is indicated above each bar/boc (C,G,I). Scale bars represent 10 μm.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Control, Staining, Binding Assay, Expressing, Inhibition, Labeling

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Schematic depicting the primary structure of full-length human MRCKβ and its functional domains. C1: protein kinase C conserved region 1. PH: pleckstrin homology. CH: citron homology. CRIB: Cdc42/Rac interactive binding. (B) Schematic outlining the different constructs generated through stepwise truncation to obtain a Cdc42·GTP biosensor, using full-length MRCKβ-GFP as a template. All constructs were expressed in MII oocytes via cRNA injection, and examined for polarized localization in the cortex overlying maternal chromosomes. The total number of oocytes scored is indicated in parentheses for each construct, with an indication of cortical polarization (+) or absence of (-). While the CRIB domain alone was not sufficient for cortical localization in vivo, stepwise extensions toward the N-terminus allowed us to define a minimal Cdc42·GTP-binding fragment encompassing the PH, CH and CRIB domains. In this study, this minimal fragment was used as a biosensor for Cdc42 activation, designated as MRCK-derived Cdc42·GTP Biosensor (MCB). Mutation of the two key histidine residues in the CRIB domain to alanine, corresponding to H1593/1596A in the full-length sequence, and indicated by a double asterisks (**), abolished polarized localization. aa: amino acids. (C) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor. (D) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor, and treated with ML-141 (5 μM, 1h). (E) Confocal image of a live MII oocyte expressing the MCB(2A)-EGFP biosensor, bearing the H1593/1596A substitution. Chromosomes were stained with SiR-DNA.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Functional Assay, Binding Assay, Construct, Generated, Injection, In Vivo, Activation Assay, Derivative Assay, Mutagenesis, Sequencing, Expressing, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Box plot showing the width of the actin cap in control MII oocytes treated with DMSO and MII oocytes treated with BDP-9066 (1 μM). P value was calculated using Student’s t -test. The number of oocytes scored is indicated above each box. (B) Immunofluorescence detection of activated myosin II (P-MRLC) and the Arp2/3 complex (p34ARC) in MII oocytes treated for 1 h with DMSO (top row) or BDP-9066 (1 μM; bottom row). White arrows point to the co-localization of P-MRLC and p34ARC at the shoulders of the actin cap. (C) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in MII oocytes treated for 1 h with DMSO (top row) or BDP-9066 (1 μM; bottom row). (D) Detection of Cdc42·GTP in a MII oocyte expressing the Cdc42 biosensor MCB-EGFP. (E) Immunofluorescence detection of MRCKβ and F-actin in MII oocytes treated for 3 h with 100 μM CK-666. In (B-E), fluorescence intensity profiles are shown, corresponding to the polarized cortex region delinated by the two grey arrowheads in the merge images. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3. Scale bars represent 10 μm.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Control, Immunofluorescence, Expressing, Fluorescence, Labeling, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Detection of Cdc42·GTP (green) and RhoA·GTP (red) in live oocytes at the metaphase-II (MII, left) and anaphase-II (AII, right) stages. Oocytes were injected at the MII stage with cRNAs encoding the MCB-EGFP and mcherry-AHPH biosensors. Images are Z-compressions of 3 consecutive confocal frames. (B) Immunofluorescence detection of MRCKβ in fixed MII (left) and AII (right) oocytes. (C) Immunofluorescence detection of ROCK1 in an AII oocyte. (D) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in an AII oocyte. (E) Immunofluorescence detection of activated myosin II (P-MRLC) in the cortex of activated oocytes undergoing anaphase II. Oocytes were treated with DMSO (top row), BDP-9066 (middle row) or a combination of BDP-9066 and Y-27632 (bottom row). Open arrowheads point to the P-MRLC rings overlying the chromatid clusters in the control (DMSO) oocyte. White arrows point to cytokinetic P-MRLC in the control (DMSO) and BDP-9066-treated oocytes. Images are z-compression of 33 (DMSO), 15 (BDP-9066) and 30 (BDP-9066 + Y-27632) consecutive confocal frames. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with SiR-DNA (panel A) or TO-PRO-3 (panels BE). Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. In (D), the white arrow points to the ingressing furrow region that is enriched in P-MRLC but devoided of MRCKβ.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Injection, Immunofluorescence, Control, Labeling, Staining, Fluorescence

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in fertilized oocytes. Top row: control oocyte treated with DMSO. Bottom row: oocyte fertilized in the presence of BDP-9066. Note the P-MRLC ring at the base of the fertilization cone (FC) in the control oocyte, and absence of in the MRCK-inhibited oocyte. Oocytes were fixed 3 hours postinsemination. PB2: second polar body. Sp: sperm chromatin. Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. (B) Bar graph depicting the percentage of fertilized oocytes showing a P-MRLC ring at the base of the FC. The fertilization medium was supplemented with DMSO (control) or BDP-9066. P value was calculated using Fisher’s exact test. (C) Schematic illustrating the experimental protocol for monitoring male pronucleus migration. (D) Detection of male and female pronuclei in zygotes fixed 8h after insemination. The left image shows a control zygote treated with DMSO after PB2 emission. The yellow line denotes the distance (d) of the male pronucleus to the nearest cortex. The right image shows a zygote treated with BDP-9066 after PB2 emission. Note the expanded male pronucleus still apposed to the FC cortex. Images are z-compressions of two consecutive confocal frames. (E) Scatter plot of the male pronucleus distance to the nearest cortex, as depicted in (D). Red and blue horizontal bars show the median values. P value was calculated using a two-tailed Mann-Whitney U test. DNA was labeled with TO-PRO-3. F-actin was labeled with Alexa Fluor 568-phalloidin. Female pronuclei were immuno-stained for H3K4me3 (green).

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Control, Fluorescence, Migration, Two Tailed Test, MANN-WHITNEY, Labeling, Staining

Journal: bioRxiv

Article Title: MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

doi: 10.1101/2022.09.25.509421

Figure Lengend Snippet: (A) Immunofluorescence detection of activated myosin II (P-MRLC) in a fertilized oocyte. Images are z-compressions across the entire zygote volume, for 3d rendition, highlighting the FC area. (B) Detection of Cdc42·GTP in the FC. The image shows a fertilized oocyte expressing the Cdc42·GTP biosensor MCB-EGFP. The oocyte was fertilized in vitro after zona pellucida removal. (C) Immunofluorescence detection of MRCKβ in a fertilized oocyte. Images are z-compressions across the entire zygote volume, for 3d rendition, highlighting the FC area. (D) Immunofluorescence detection of activated myosin II (P-MRLC) and MRCKβ in a fertilized oocyte. Fluorescence intensity profiles correspond to the cortical regions delineated by the grey arrowheads in the merge images. FC: fertilization cone. Sp: sperm chromatin. PB2: second polar body. F-actin was labeled with Alexa Fluor 568-phalloidin. DNA was stained with TO-PRO-3.

Article Snippet: The following primary antibodies were used : Phospho-myosin light chain 2 (Ser19) (1:200; Cell Signaling Technology #3671), Non-muscle myosin IIA (1:100; Abcam ab24762), Myosin IIB (D8H8) (1:100; Cell Signaling Technology #8824), MRCKα (B-3) (1:100; Santa Cruz Biotechnology sc-374568), MRCKβ (A-2) (1:100; Santa Cruz Biotechnology sc-390127), Tubulin (1:200; Abcam ab6161), p34-ARC (1:100; Santa Cruz Biotechnology sc-515754), Rock-1 (K-18) (1:100; Santa Cruz Biotechnology sc-6056), Trimethylhistone H3 (Lys4) (1:100; Merck Millipore 04-745).

Techniques: Immunofluorescence, Expressing, In Vitro, Fluorescence, Labeling, Staining

Journal: Cancer research

Article Title: Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer

doi: 10.1158/0008-5472.CAN-17-2870

Figure Lengend Snippet: (A) Structures of 7-azaindole-3-carbonitrile hit fragment, BD8900 and BDP9066. (B) BDP8900 dose-response curves for inhibition of MRCKα, MRCKβ, ROCK1 and ROCK2 kinase activity in vitro at 1 µM ATP. (C) BDP9066 dose-response curves for inhibition of MRCKα, MRCKβ, ROCK1 and ROCK2 kinase activity in vitro at 1 µM ATP. Results shown are mean ± SD of duplicate independent replicates. (D) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinase domains were treated with BDP8900 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. Inhibition of MLC2 phosphorylation by BDP8900 for each induced kinase domain. Results shown are mean ± SEM of 3-4 independent replicates. (E) Cells expressing doxycycline-induced MRCKβ, ROCK1 or ROCK2 kinase domains were treated with BDP9066 at indicated concentrations for 60 minutes prior to lysis and quantitative western blotting. Inhibition of MLC2 phosphorylation by BDP8900 for each induced kinase domain. Results shown are mean ± SEM of 3-4 independent replicates.

Article Snippet: The following antibodies were used: rabbit anti-MRCKα pS1003 antibody raised by Eurogentec using the peptide AcNH -KGCPG-S ( PO 3 H 2) -TGFPP- CONH2 , rabbit anti-pMLC2 Thr18/Ser19 (#3674; Cell Signaling Technology), mouse anti-MRCL3/MRLC2/MYL9 (sc-28329; Santa Cruz Biotechnology), mouse anti α-tubulin (T9026; Sigma), rabbit anti-ERK2 (Chris Marshall, Institute of Cancer Research, London UK), mouse anti-MRCKα (H00008476-M01; Abnova), mouse anti-MRCKβ (H00009578-M03; Abnova), mouse anti-MRCKαβ (MANDM1 6G8; Glenn Morris, Centre for Inherited Neuromuscular Disease, Oswestry UK) ( 23 ), mouse anti-ROCK1 (BD-611136; BD Biosciences), mouse anti-ROCK2 (BD-61062; BD Biosciences), mouse anti-Cas9 (C15200229; Diagenode), mouse anti-FLAG (F4042; Sigma).

Techniques: Inhibition, Activity Assay, In Vitro, Expressing, Lysis, Western Blot

Journal: Cancer research

Article Title: Discovery of potent and selective MRCK inhibitors with therapeutic effect on skin cancer

doi: 10.1158/0008-5472.CAN-17-2870

Figure Lengend Snippet: (A) Stained SDS-PAGE gel (left) of FLAG immunoprecipitated FLAG-MRCKα and FLAG-MRCKα K106M after in vitro 32P incorporation assay. Arrow indicates MRCKα. Gel autoradiograph (right) shows 32P labelling of MRCKα. MW = molecular weight. (B). Stained SDS-PAGE gel (left) of FLAG-immunoprecipitates from control HEK293 cells, or cells transfected with pEF-FLAG-MRCKα or pEF-FLAG-MRCKα K106M. Arrow indicates MRCKα. MW = molecular weight. Higher energy collision dissociation (HCD) MS/MS fragmentation spectra (right) of MRCKα tryptic peptide 999-1009 containing S1003 (red lettering) in MRCKα or MRCKα K106M (phosphorylation inferred from y7, y7-H3PO4 and y6 signals indicated with green arrows). See Supplemental Table 8 for theoretical y and b fragmentation ion series masses. Diagram of MRCKα protein domains (inset) illustrating location of S1003. (KD = kinase domain, CC = Coiled-coiled domain, C1 = C1 diacylglycerol binding domain, PH = Pleckstrin homology domain, CH = Citron Homology domain, CRIB = Cdc42/Rac interactive binding motif). (C). Dot blot of MRCKα peptides (10 to 500 pg) containing phosphorylated S1003 (peptide P) or unphosphorylated S1003 (peptide U), stained with pS1003 antibody (D). Western blots of FLAG-immunoprecipitated FLAG-MRCKα stained with untreated pS1003 antibody, or pS1003 antibody pre-incubated with phosphorylated S1003 peptide P or unphosphorylated peptide U (E). Western blots of FLAG-immunoprecipitated FLAG-MRCKα, incubated in phosphatase buffer without (control) or with lambda phosphatase (phosphatase) (F). Western blot of FLAG-immunoprecipitated FLAG-MRCKα, FLAG-MRCKα K106M or FLAG-MRCKα S1003A. (G). Western blot (left) of FLAG-immunoprecipitated FLAG-MRCKα incubated in kinase buffer in the absence or presence of recombinant CDC42 Q61L protein. Relative S1003 phosphorylation revealed that CDC42 significantly increased autophosphorylation. Results shown are means ± SEM from 3 experiments. Student’s t-test (*** = p<0.001). (H). Western blots of HEK293 cells expressing FLAG-MRCKα treated with DMSO, 3 µM BDP5290 or 1 µM BDP9066 (I). Western blots of FLAG immunoprecipitated FLAG-MRCKαΔC or FLAG-MRCKα K106M untreated or treated with lambda phosphatase, or treated with lambda phosphatase and then incubated in kinase buffer to allow autophosphorylation (AutoPhos). (J) Western blots of FLAG-immunoprecipitated FLAG-MRCKα, untreated or treated with lambda phosphatase (upper panel). Incubation with kinase buffer resulted in autophosphorylation (pS1003; lower left panel) and recombinant MLC2 substrate phosphorylation (pMLC2; lower right panel) (K). Western blots of FLAG-immunoprecipitated FLAG-MRCKα, FLAG-MRCKα K106M or FLAG-MRCKα S1003A incubated in vitro with recombinant MLC2 in a kinase buffer (left). Relative MLC2 phosphorylation by each kinase revealed that K106M significantly reduced activity, while S1003A was not different from wild-type. Results shown are means ± SEM from 5 independent experiments. One way Kruskal-Wallis ANOVA followed by post-hoc Dunn’s multiple comparison (* = p<0.05). (L) Relative MRCKα (upper) and MRCKβ (lower) expression was significantly higher in cutaneous SCC relative to normal human skin determined by microarray analysis (45). Results shown are means ± SD from 6 normal skin and 5 SCC patients. Two-tailed Mann-Whitney test of significance (** = p<0.01). (M) Immunohistochemistry of MRCKα, MRCKα pS1003 or MRCKβ in benign papillomas from mice expressing oncogenic Hras in epidermal keratinocytes, or in well-differentiated squamous cell carcinomas resulting from Hras/c-Fos/Pten-/- genetic modifications or DMBA/TPA chemical carcinogenesis. Scale bars = 100 µm.

Article Snippet: The following antibodies were used: rabbit anti-MRCKα pS1003 antibody raised by Eurogentec using the peptide AcNH -KGCPG-S ( PO 3 H 2) -TGFPP- CONH2 , rabbit anti-pMLC2 Thr18/Ser19 (#3674; Cell Signaling Technology), mouse anti-MRCL3/MRLC2/MYL9 (sc-28329; Santa Cruz Biotechnology), mouse anti α-tubulin (T9026; Sigma), rabbit anti-ERK2 (Chris Marshall, Institute of Cancer Research, London UK), mouse anti-MRCKα (H00008476-M01; Abnova), mouse anti-MRCKβ (H00009578-M03; Abnova), mouse anti-MRCKαβ (MANDM1 6G8; Glenn Morris, Centre for Inherited Neuromuscular Disease, Oswestry UK) ( 23 ), mouse anti-ROCK1 (BD-611136; BD Biosciences), mouse anti-ROCK2 (BD-61062; BD Biosciences), mouse anti-Cas9 (C15200229; Diagenode), mouse anti-FLAG (F4042; Sigma).

Techniques: Staining, SDS Page, Immunoprecipitation, In Vitro, Autoradiography, Molecular Weight, Transfection, Tandem Mass Spectroscopy, Binding Assay, Dot Blot, Western Blot, Incubation, Recombinant, Expressing, Activity Assay, Microarray, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry