Journal: Nature structural & molecular biology

Article Title: Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II

doi: 10.1038/nsmb.2399

Figure Lengend Snippet: ChIP of Pol II, DSIF (Spt5), NELF and TFIIEα on: ( a ) GAPDH ; ( b ) c -Myc ; and ( c ) p21 genes. Treatment with 50 μM DRB results in increased Spt5 and Pol II crosslinking over the TSS, whereas inhibition of Cdk7 in Cdk7 as cells with 10 μM 3-MP-PP1 results in reduced Pol II pausing and a loss of Spt5 and NELF recruitment, which is accompanied by an increase in TFIIE crosslinking ( n =2-4, P <0.01; ). ( d ) Change in the ratio of Spt5 and TFIIE relative to Pol II after drug treatments. The data, averaged from four genes ( GAPDH , c-Myc , p21 and CHD2 ), were normalized to the Spt5- and TFIIE-to-Pol II ratios in the DMSO-treated condition, which were each set at 1. This relationship varied little among the tested genes , suggesting a generalized anti-correlation between Pol II-associated DSIF and TFIIE.

Article Snippet: We obtained antibodies to Cdk9 (sc-8338), cyclin T1 (sc-10750), RNA Pol II (sc-899 and sc-9001), Spt5 (sc-28678), TFIIEα (sc-237) and NELF-A (sc-23599) from Santa Cruz Biotechnology; RNA Pol II phosphorylated at Ser2 (A300-654A) or Ser5 (A300-655A) from Bethyl Laboratories; histone H2B (07-371) and H2BUb1 (05-1312) from Millipore; Cdk9-T186P (2549), used for immunoblots only, from Cell Signaling Research; and Cdk7 (MO1.1) from Sigma.

Techniques: Inhibition

Journal: Nature structural & molecular biology

Article Title: Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II

doi: 10.1038/nsmb.2399

Figure Lengend Snippet: ( a ) Kinase assay of WT and AS Cdk9. Cdk9 as —cyclin T1, purified as a complex from insect cells co-infected with the corresponding baculoviruses, prefers the bulky analog N6 (benzyl)-ATP over natural ATP to radiolabel GST-Spt5, whereas wild-type Cdk9—cyclin T1 (purified identically) prefers natural ATP. ( b ) Assay of Cdk9 as kinase activity following incubation with activating kinases. Unphosphorylated Cdk9 as complexed with cyclin T1 was incubated with either Cdk7 or Csk1, resulting in increased activity towards GST-Spt5. ( c ) Phosphorylation with radiolabeled N6 -(benzyl)-ATP of HeLa nuclear extract or purified fractions of DSIF and Pol II reveals activation of Cdk9 as —cyclin T1 by Cdk7. The labeling in nuclear extract demonstrates the CAK-dependent phosphorylation by Cdk9 as of a limited number of substrates, several of which remain to be identified. ( d ) Activation of Cdk9 as by cell extracts. Cdk9 as —cyclin T1 was incubated with ATP and whole cell extract—mock- or Cdk7-immunodepleted—prior to testing its activity on a recombinant GST-Spt5 substrate . Depletion of Cdk7 reduces the ability of the extract to activate Cdk9 as , indicating the removal of a Cdk9-activating kinase. ( e ) Activation of Cdk9 as by cell extracts. Treatment with 200 nM 3-MB-PP1 , diminishes the Cdk9 as -activating kinase activity in Cdk7 as but not wild-type HCT1116 extracts; activity is restored to the inhibited extract by the addition of purified, inhibitor-resistant, wild-type Cdk7 (Cdk7 WT ).

Article Snippet: We obtained antibodies to Cdk9 (sc-8338), cyclin T1 (sc-10750), RNA Pol II (sc-899 and sc-9001), Spt5 (sc-28678), TFIIEα (sc-237) and NELF-A (sc-23599) from Santa Cruz Biotechnology; RNA Pol II phosphorylated at Ser2 (A300-654A) or Ser5 (A300-655A) from Bethyl Laboratories; histone H2B (07-371) and H2BUb1 (05-1312) from Millipore; Cdk9-T186P (2549), used for immunoblots only, from Cell Signaling Research; and Cdk7 (MO1.1) from Sigma.

Techniques: Kinase Assay, Purification, Infection, Activity Assay, Incubation, Activation Assay, Labeling, Recombinant

Journal: Nature structural & molecular biology

Article Title: Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II

doi: 10.1038/nsmb.2399

Figure Lengend Snippet: ( a ) ChIP of RNA polymerase II (Pol II), total Cdk9, and Cdk9 phosphorylated on Thr186 at different positions along the c-Myc gene. The Thr186-phosphorylated form of Cdk9 is enriched towards the 3′ end of the gene and is diminished by addition of 3-MB-PP1 (10 μM) to Cdk7 as cells. ( b ) Same as in ( a ), for the GAPDH gene.

Article Snippet: We obtained antibodies to Cdk9 (sc-8338), cyclin T1 (sc-10750), RNA Pol II (sc-899 and sc-9001), Spt5 (sc-28678), TFIIEα (sc-237) and NELF-A (sc-23599) from Santa Cruz Biotechnology; RNA Pol II phosphorylated at Ser2 (A300-654A) or Ser5 (A300-655A) from Bethyl Laboratories; histone H2B (07-371) and H2BUb1 (05-1312) from Millipore; Cdk9-T186P (2549), used for immunoblots only, from Cell Signaling Research; and Cdk7 (MO1.1) from Sigma.

Techniques:

Journal: Nature structural & molecular biology

Article Title: Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II

doi: 10.1038/nsmb.2399

Figure Lengend Snippet: ChIP analysis of Pol II on ( a ) c-Myc and ( b ) GAPDH genes, showing the effect of Cdk7 inhibition on the relative levels of CTD phosphorylation at Ser2 and Ser5. When expressed as a ratio to total Pol II, Ser5 phosphorylation (Ser5P) is reduced at the 5′ ends of both genes. Levels of Ser2 phosphorylation (Ser2P) are reduced relative to total Pol II at the 3′ ends of both genes, where Cdk9-Thr186P and Ser2P signals are highest in mock-treated cells ( n =4, error bars: s.d., P vaues: student's t -test). ( c ) Simultaneous inhibition of Cdk7 and Cdk9 has synergistic effects on bulk Pol II phosphorylation. Single-kinase inhibition of Cdk7 as with 10 μM 3-MB-PP1 or of Cdk9 with 50 μM DRB or 100 nM FP had modest or no effects on levels of Ser2P, Ser5P or the hyperphosphorylated II 0 form of Rpb1, whereas combined Cdk7 and Cdk9 inhibition reduced all three signals.

Article Snippet: We obtained antibodies to Cdk9 (sc-8338), cyclin T1 (sc-10750), RNA Pol II (sc-899 and sc-9001), Spt5 (sc-28678), TFIIEα (sc-237) and NELF-A (sc-23599) from Santa Cruz Biotechnology; RNA Pol II phosphorylated at Ser2 (A300-654A) or Ser5 (A300-655A) from Bethyl Laboratories; histone H2B (07-371) and H2BUb1 (05-1312) from Millipore; Cdk9-T186P (2549), used for immunoblots only, from Cell Signaling Research; and Cdk7 (MO1.1) from Sigma.

Techniques: Inhibition

Journal: Nature structural & molecular biology

Article Title: Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II

doi: 10.1038/nsmb.2399

Figure Lengend Snippet: ( a ) Activity assay of Cdk7. Efficient phosphorylation of TFIIEα and -β by Cdk7 depends on both T-loop phosphorylation of Cdk7 and presence of Mat1, as in the case of the Pol II CTD. Cdk7 with or without T-loop phosphorylation is denoted by P-Cdk7 or Cdk7, respectively, cyclin H by H, and Mat1 by M. (Arrow points to labeled TFIIEβ; asterisk denotes Mat1 phosphorylated by associated Cdk7 as ). ( b ) Cdk9 phosphorylation by Cdk7. Cdk9, like Cdk2, is phosphorylated with similar efficiency by all isoforms of Cdk7. T-loop phosphorylation of Cdk9 as was detected by immunoblotting with anti-P-Thr186. ( c ) ChIP analysis of Cdk7. Total Cdk7 signals are enriched in the promoter-proximal region, whereas T-loop phosphorylation increases from the 5′ to 3′ end of the c-Myc gene. ( d ) The ratio of T-loop phosphorylated: total Cdk7 increases along the length of c-Myc . ( e) Proposed model of CDK-dependent, promoter-proximal pause establishment and release: 1. After recruitment of TFIIE and TFIIH (Cdk7), Cdk7 activity allows TFIIE to be dislodged by DSIF from the Poll II clamp. The involvement of TFIIE phosphorylation by Cdk7 (gray dashed arrow) in this switch remains hypothetical, and other phosphorylations catalyzed by Cdk7 could be implicated. Binding of DSIF and NELF blocks elongation (pause). 2. Cdk9 is activated by Cdk7, and 3. phosphorylates DSIF and NELF to trigger processive elongation (release). Inset: Cdk7 sits atop a feed-forward loop; its catalytic activity is required to initiate Pol II pausing through the TFIIE-DSIF switch and to release the pause through activation of Cdk9.

Article Snippet: We obtained antibodies to Cdk9 (sc-8338), cyclin T1 (sc-10750), RNA Pol II (sc-899 and sc-9001), Spt5 (sc-28678), TFIIEα (sc-237) and NELF-A (sc-23599) from Santa Cruz Biotechnology; RNA Pol II phosphorylated at Ser2 (A300-654A) or Ser5 (A300-655A) from Bethyl Laboratories; histone H2B (07-371) and H2BUb1 (05-1312) from Millipore; Cdk9-T186P (2549), used for immunoblots only, from Cell Signaling Research; and Cdk7 (MO1.1) from Sigma.

Techniques: Activity Assay, Labeling, Western Blot, Binding Assay, Activation Assay

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) C33A cells expressing EV, HCD13 or Y6F were treated with vehicle (Veh) or aluminum fluoride (AlF; 50 μM) for 30 min, and membrane fractions were purified from total cell lysates. Active ARF6-GTP in the fractions was measured by pull down assay and normalized to total ARF6. Validation of the membrane fraction was verified by expression of plasma membrane marker E-cadherin. (B) Plots depict quantification of immunoblot analysis of ARF6-GTP, CD13, and IQGAP1 normalized to E-cadherin in the plasma membrane. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Purification, Pull Down Assay, Marker, Western Blot, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) Lysates from C33A cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1 and total ARF6 by immunoblot analysis. ARF6-GTP was detected by a pull-down assay of the immunoprecipitates using beads conjugated to the PBD binding domain of GGA3 for 1 hour at 4°C. (B) Lysates from T27N ARF6 dominant-negative mutant cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1, ARF6 or HA-tag by immunoblot analysis. Active ARF6 was detected by pull down with GGA3-conjugated beads from the immunoprecipitates. (C and D) ARF6 activity was measured in C33A cells expressing EV or HCDi3 after cell-ECM adhesion over the indicated time. Using ARF6 protein-binding domain (PBD) of the effector protein GGA3 conjugated beads, which specifically binds the GTP-bound form of ARF6, the subsequent pull-down of ARF6-GTP was quantified by immunoblot analysis using the ARF6-specific antibody. Blots (C) are representative and quantified data (D) are means ± SD of 3 independent experiments. (E and F) β1-integrin receptor recycling was measured in a pulse-chase assay with β1 integrin Ab clone 12G10 (for C33A cells; E) or 9EG7 (for MEFs; F) in the presence of N-myristolated ARF6 inhibitor peptide. Serum-starved cells were treated with β1-integrin Ab at 4°C for 30 min, pulsed for 1 hour to induce internalization, acid-stripped, washed, treated with myr-ARF6 peptide (10 μM) for 30 min, and allowed to recycle at 37°C for 2 to 4 hours. Paraformaldehyde-fixed cells were stained with fluorescently conjugated secondary Ab and MFI (Mean Fluorescence Intensity) of surface β1-integrin was analyzed by flow cytometry. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.0i by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Binding Assay, Dominant Negative Mutation, Activity Assay, Protein Binding, Pulse Chase, Staining, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A and B) In a scratch assay, following injury on the monolayer by creating a scratch, C33A cells expressing HCD13 were allowed to migrate to the wound and fixed with 4% paraformaldehyde at the 6-hour time point. Cells were stained with phalloidin (red; left rows) or CD13 (red; right rows) and IQGAP1 (green) and imaged using confocal microcopy; magnified inset of CD13/IQGAP1-stained C33A-HCD13 cells is shown (B). Scale bar; 5μm. DAPI (blue). (C) Quantification of the area of F-actin and IQGAP1 accumulation at the migrating front of the cell, represented in (A), normalized to total cell area by Fiji software. Five fields were counted for each genotype, and all cells in each field were measured. (D) Percent of F-actin+, CD13+ and IQGAP1+ cells from (A) at the leading front were measured in each of five fields for each genotype. Data are mean ± SD of 3 independent experiments. *P<0.05 by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Wound Healing Assay, Expressing, Staining, Software, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: In wild-type cells, phospho-CD13 and β1-integrin internalize into early endosomes, sort to recycling endosomes and return to the cell membrane, enabling cell-ECM adhesion and migration. However, in cells lacking CD13 or expressing an inactive CD13 mutant, whereas β1-integrin internalizes into early endosomes, it aberrantly traffics to Rab7+ lysosomes and it is ultimately degraded. Mechanistically, CD13 must be present in a complex containing the scaffolding protein IQGAP1, active-ARF6 its GEF EFA6 and β1-integrin at the plasma membrane to allow proper β1-integrin recycling and cell migration to proceed. In the absence of CD13, no active-ARF6 is detected in the plasma membrane and IQGAP1 is not recruited to the migrating front, thereby diminishing cell adhesion, spreading and migration.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Migration, Expressing, Mutagenesis, Scaffolding

Journal: Blood

Article Title: A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma

doi: 10.1182/blood-2010-04-276626

Figure Lengend Snippet: ONX 0912 triggers apoptosis in MM cells, associated with PARP cleavage and caspase activation. (A) MM cell lines were treated with ONX 0912 for 48 hours and analyzed for apoptosis using annexin V/PI staining assay. (B-C) MM.1S cells were treated with ONX 0912 at the indicated doses for 48 hours and harvested; whole-cell lysates were subjected to immunoblot analysis with anti-PARP, anti–caspase-3, anti–caspase-8, anti–caspase-9, or anti-GAPDH Abs. FL indicates full-length; CF denotes cleaved fragment. Blots shown are representative of 3 independent experiments.

Article Snippet: Membranes were blocked by incubation in 5% nonfat dry milk in PBST (0.05% Tween-20 in phosphate-buffered saline [PBS]), and probed with specific antibodies against poly(ADP) ribose polymerase (PARP; BD Biosciences Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; BD Biosciences Pharmingen), or caspase-8, caspase-9, or caspase-3 (Cell Signaling Technology).

Techniques: Activation Assay, Staining, Western Blot