G12 signaling through c-jun nh 2-terminal kinase promotes breast cancer cell invasion

10.1371/journal.pone.0026085PLoS ONE611

Characterization of the unfolding of ribonuclease A by a pulsed hydrogen exchange study: evidence for competing pathways for unfolding

The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10°C using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, N∗I, in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form N∗I on one route. On the other route an exchange-incompetent intermediate state ensemble, I2U, is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that N∗I and I2U are nativelike in their secondary and tertiary structures. While N∗I unfolds directly to the fully exchange-competent unfolded state (U), IU2 forms another intermediate IU3 which then unfolds to U. IU3 is devoid of all native α-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core

Gα12QL activates JNK in breast cancer cells.

<p>MDA-MB-231 cells were infected with GFP (control) or Gα12QL adenovirus for 6–7 h. (A) Cells were starved for 2 h and then treated for the indicated time with JNK inhibitor SP600125 (20 µM) prior to lysis in JNK activation buffer and processing for analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026085#s2" target="_blank"><i>Materials and Methods</i></a>). (B) Cells were transfected with JNK siRNA or control (luc) siRNA for ∼72 h prior to infection with adenovirus as in (A). Cells were starved for 18 h before being lysed. For both panels, equal amounts of total protein in lysates were subjected to SDS-PAGE and immunoblot analysis to assess levels of phospho-JNK (P-JNK, p54 and p46) and phospho-c-Jun (P-c-Jun). Total levels of JNK and Gα12 expression, along with α-tubulin as a loading control, are also shown. Each panel shows the results of a single experiment that is representative of three or more independent experiments.</p

NMR identification and characterization of the flexible regions in the 160 kDa molten globule-like aggregate of barstar at low pH

Barstar is known to form a molten globule-like A form below pH 4. This form exists as a soluble aggregate of 16 monomeric subunits, and appears to remain homogeneous in solution for at least two weeks. Here, structural characterization by NMR of the flexible regions in the A form of barstar has been carried out at pH 2.7 and 25 &#176;C. Significantly, the A form appears to be a symmetrical aggregate. Using the recently described fast assignment strategy from HNN and HN(C)N spectra, along with the standard triple resonance and three-dimensional NMR experiments, the flexible segment of the aggregate has been identified to belong largely to the N-terminal end of the polypeptide chain; sequential connectivities were obtained for the first 20 residues (except two) from these experiments. This segment is free in each of the monomeric subunits, and does not form a part of the aggregated core of the A form. The secondary chemical shifts of these residues suggest propensity toward an extended structure. Their 3JHN,H&#945; coupling constants have values corresponding to those in a random coil structure. However, a few medium-range NOEs, some of them involving side chain atoms, are observed between some residues in this segment. The lowered temperature coefficients of the HN chemical shifts compared to random coil values indicate possibilities of some hydrogen bonding in this region. Analysis of the 15N relaxation parameters and reduced spectral density functions, in particular the negative values of heteronuclear NOEs, indicates large-amplitude high-frequency motions in the N-terminal segments; the first three residues show more negative NOEs than the others. The 15N transverse relaxation rates and the J(0) spectral density values for residues Ser12 and Ser69 are significantly larger than for the rest, indicating some microsecond to millisecond time scale conformational exchange contributions to the relaxation of these residues. Taken all together, the data suggest that the A form of barstar is an aggregate with a rigid core, but with the N-terminal 20 residues of each of the monomeric subunits, in a highly dynamic random coil conformation which shows transient local ordering of structure. The N-terminal segment, anchored to the aggregated core, exhibits free-flight motion

Gα12QL activates a ROCK-JNK signaling axis.

<p>MDA-MB-231 cells were infected with adenovirus harboring GFP (control), Gα12QL or dominant-negative RhoA (DN RhoA) for 6–7 h. (A) Cells were starved and treated with 1 U/ml thrombin for 10 min, as indicated, before lysis in JNK activation buffer. Equal amounts of total protein in lysates were subjected to SDS-PAGE and immunoblot analysis to assess levels of phospho-JNK (P-JNK, p54 and p46) and phospho-c-Jun (P-c-Jun). Total level of JNK expression, along with Rho GDI as a loading control, is also shown. (B) Cells were starved and treated with ROCK inhibitor Y-27632 (10 µM) for 2 h before lysis in JNK activation buffer. Equal amounts of total protein in lysates were subjected to SDS-PAGE and immunoblot analysis to assess levels of phospho-JNK (P-JNK, p54 and p46) and phospho-c-Jun (P-c-Jun). Total levels of JNK and Gα12 expression, along with α-tubulin as a loading control, are also shown. Data shown in (A) and (B) are from a single experiment that is representative of three independent experiments. (C) Cells infected with GFP or Gα12QL adenovirus were starved for ∼18 h, then pre-treated with Y-27632 (10 µM) for 2 h and allowed to invade Matrigel for 24 h in the presence of the inhibitor, towards 5 µg/ml fibronectin. Cells that invaded were stained and counted in four random optical fields for each transwell, with three transwells per condition. Results are expressed as fold change in invasion compared to vehicle-treated GFP control cells. The <i>inset</i> shows immunoblot analysis of Gα12 and α-tubulin (loading control) levels. Data is presented as mean ± SE from a single experiment that is representative of three independent experiments. *** <i>p</i><0.001, as determined by one-way ANOVA, followed by Tukey's multiple comparison test to obtain <i>p</i> values.</p

ROCK and JNK signal downstream of Gα12QL-activated Rho.

<p>(A) MDA-MB-231 cells were transfected with control (luc) siRNA or JNK siRNA for 3 days prior to infection with GFP (control) or Gα12QL adenovirus for 6–7 h. Cells were starved for ∼18 h and lysed. Lysates were subjected to pull-down assays using a GST fusion construct of the activated RhoA-binding domain of rhotekin, and levels of precipitated Rho-GTP were determined by immunoblot analysis using anti-RhoA antibody. Total levels of RhoA, Gα12 and JNK in the lysates are also shown. (B) The data in the anti-Rho immunoblots shown in (A) was quantified by plotting the intensities as percentage of activated Rho to total Rho for each sample. (C) MDA-MB-231 cells were plated on coverslips and transfected with siRNA as stated above, prior to infection with GFP (control) (<i>panels a</i>, <i>d</i>) or Gα12QL (<i>panels b</i>, <i>e</i>) adenovirus. Cells were starved for ∼18 h, and those in two conditions (<i>panels c</i>, <i>f</i>) treated with 1 U/ml thrombin for 10 min as indicated before all cells were fixed and stained with rhodamine-phalloidin to visualize actin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026085#s2" target="_blank"><i>Materials and Methods</i></a>). Representative images for each condition are shown. Bar, 50 µM. Each panel shows the results of a single experiment that is representative of three independent experiments. (D) Fluorescence intensities of rhodamine-phalloidin staining were quantified using MetaMorph 7.5 software. Average fluorescence intensities are plotted as mean ± SE for data from two slides imaged with the same exposure time for every condition shown; ∼220 cells (<i>panels a</i>, <i>c</i>), and ∼120 cells (<i>panel b</i>) quantified per slide.</p

JNK is activated downstream of G12 signaling in 4T1 mammary carcinoma cells and is required for cell invasion.

<p>(A) 4T1 cells were infected with adenovirus harboring GFP (control) or the RGS domain of p115RhoGEF (p115RGS) for ∼20 h, and then starved for 8 h. Cells were allowed to invade Matrigel for 24 h in the presence of thrombin (1 U/ml), towards 3% FBS. Cells that invaded were stained and counted in four random optical fields for each transwell, with three transwells per condition. Results are shown as percent invasion compared to invasion in the control cells. The <i>inset</i> shows p115RGS expression. Data is presented as mean ± SE from a single experiment that is representative of three independent experiments. * <i>p</i><0.05 as determined by one-way ANOVA, followed by Tukey's multiple comparison test to obtain <i>p</i> values. (B) 4T1 cells were infected with adenovirus harboring GFP (control) or p115RGS, as indicated, for ∼18–19 h and starved for ∼9 h. Cells were then treated with 1 U/ml thrombin for 10 min, as indicated, before lysis in JNK activation buffer. Equal amounts of total protein in the lysates were separated by SDS-PAGE and immunoblot analysis was performed to assess levels of phospho-JNK (P-JNK, p54 and p46) and phospho-c-Jun (P-c-Jun). Total levels of JNK, myc-p115RGS and RhoGDI (loading control) are also shown. Data shown are from a single experiment that is representative of three independent experiments. (C) 4T1 cells were transfected with control (luc) and JNK siRNA for 67–68 h, and then starved for 8 h. Cells were allowed to invade Matrigel for 24 h in the presence of thrombin, towards 3% FBS. Cells that invaded were quantified as detailed for panel A. Results are shown as percent invasion compared to invasion in the control cells. The <i>inset</i> shows immunoblot analysis of JNK and Rho GDI (loading control) levels. Data is presented as mean ± SE from a single experiment that is representative of three independent experiments. ** <i>p</i><0.01 as determined by one-way ANOVA, followed by Tukey's multiple comparison test.</p

The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10°C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (Tr≈1 ns) local motion of its indole side-chain, and (ii) a slower (Tr≈7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(±0.01) min−1 This rate compares to a rate of folding of 0.03(±0.005) min−1, determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min−1, when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5.6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(±0.06) min−1. These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein