Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics

c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck

Separation of Anti-Proliferation and Anti-Apoptotic Functions of Retinoblastoma Protein through Targeted Mutations of Its A/B Domain

BACKGROUND: The human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif. METHODOLOGY/PRINCIPLE FINDINGS: Based on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis. CONCLUSION/SIGNIFICANCE: Previous studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Platelet-Derived Growth Factor C Is Upregulated in Human Uterine Fibroids and Regulates Uterine Smooth Muscle Cell Growth1

Leiomyomata uteri (i.e., uterine fibroids) are benign tumors arising from the abnormal growth of uterine smooth muscle cells (SMCs). We show here that the expression of platelet-derived growth factor C (PDGFC) is higher in approximately 80% of uterine fibroids than in adjacent myometrial tissues examined. Increased expression of PDGFC is also observed in fibroid-derived SMCs (fSMCs) relative to myometrial-derived SMCs (mSMCs). Recombinant bioactive PDGFCC homodimer stimulates the growth of fSMCs and mSMCs in ex vivo cultures and prolongs the survival of fSMCs in Matrigel plugs implemented subcutaneously in immunocompromised mice. The knockdown of PDGF receptor-alpha (PDGFRA) through lentiviral-mediated RNA interference reduces the growth of fSMCs and mSMCs in ex vivo cultures and in Matrigel implants. Furthermore, two small molecule inhibitors of the PDGFR tyrosine kinase (i.e., imatinib and dasatinib) exerted negative effects on fSMC and mSMC growth in ex vivo cultures, albeit at concentrations that cannot be achieved in vivo. These results suggest that the PDGFCC/PDGFRA signaling module plays an important role in fSMC and mSMC growth, and that the upregulation of PDGFC expression may contribute to the clonal expansion of fSMCs in the development of uterine fibroids

A) RB phosphorylation by Cdk2/Cyclin E in SAOS-2 cells.

<div><p>Cells were infected with recombinant adenovirus expressing RB or RB-KN, in the absence or presence of co-infection with recombinant adenovirus expressing Cdk2 and Cyclin E.</p> <p>The electrophoresis mobility of RB was examined by immunoblotting of whole cell lysates with anti-RB.</p> <p>B) Representative FACS profiles. SAOS-2 cells infected with the indicated recombinant adenoviruses were treated with 50 µM etoposide, harvested 48 hours later, fixed, stained with propidium iodide and the DNA content analyzed by FACS.</p> <p>The gate for sub-G1 DNA fraction is shown in each profile.</p> <p>C) Summary of DNA fragmentation results.</p> <p>The sub-G1 DNA content was determined by FACS analysis as illustrated in panel B.</p> <p>The values are mean and standard errors from three independent experiments.</p></div

A) BrdU incorporation.

<div><p>MDA-MB468 cells were infected with the indicated recombinant adenoviruses encoding each of the indicated proteins.</p> <p>Infected cells were allowed to incorporate BrdU over a period of 14 hours before they were fixed and stained with phycoerythrin (PE)-conjugated monoclonal antibody against BrdU.</p> <p>The fraction of BrdU-positive cells in each sample was measured by flow cytometry (FACS).</p> <p>The levels of BrdU incorporation in GFP-virus and RB-KN-virus infected cells were statistically similar by student <i>t</i>-test (*) from three independent experiments.</p> <p>Representative FACS profiles with the BrdU-positive gates shown are displayed to the right of the histogram.</p> <p>B) Flat cell formation. SAOS-2 cells transfected with plasmids expressing neomycin resistance and each of the indicated RB proteins were cultured in G418 for two weeks.</p> <p>The giant flat cells were stained with crystal violet and their numbers counted under a dissection microscope.</p> <p>The number of flat cells induced by RB in each of four independent experiments was set at 100%.</p> <p>The means and standard errors of relative flat cell induced by vector, RB-K, RB-N and RB-KN from four experiments are shown.</p> <p>Student <i>t</i>-test showed the number of flat cells found in RB-KN-transfected cultures was significantly different (* p<0.002) from that in RB-transfected cultures.</p></div

A) Summary of RB mutants.

<div><p>The amino acid numbering is that of the human retinoblastoma protein.</p> <p>Two shaded boxes represent the A and B domains; I, the insert region; N, the region N-terminal to the A domain; C, the region C-terminal to the B domain.</p> <p>B) GST pull-down assay.</p> <p>The RB-deficient MDA-MB468 cells were infected with recombinant adenovirus encoding GFP, RB, RB-K, RB-N or RB-KN, whole cell lysates were incubated with purified GST-E2F1 or GST-E7 fusion protein immobilized on glutathione-Sepharose.</p> <p>The bound fractions were solubilized, fractionated by SDS-PAGE and reacted with an affinity-purified anti-RB.</p> <p>C) Reciprocal GST pull-down assay.</p> <p>Lysates from SAOS-2-tet-E2F1 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Phillips1" target="_blank">[21]</a> were immunoblotted with anti-E2F1 to demonstrate the induced expression of E2F1 protein (upper panel).</p> <p>Lysates from induced and non-induced cells were incubated with GST-RB or GST-RB-K fusion proteins immobilized on glutathione-Sepharose, and the bound fraction analyzed by immunoblotting with anti-E2F1 (lower panel).</p> <p>D) Co-immunoprecipitation assay.</p> <p>SAOS-2-tet-E2F1 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Phillips1" target="_blank">[21]</a> were infected with recombinant adenoviruses encoding GFP, RB, RB-K, RB-N or RB-KN and then cultured without tetracycline to induce the expression of E2F1.</p> <p>Co-immunoprecipitation of E2F1 in anti-RB immune complex was determined by immunblotting with anti-E2F1 antibody (α-E2F1). E) Luciferase assay.</p> <p>The Gal4-Luc reporter plasmid contains a firefly luciferase expression cassette with five Gal4 binding sequence motifs.</p> <p>The Gal4-E2F1 protein contains the Gal4-DNA binding domain and the transactivation domain of E2F1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Flemington1" target="_blank">[22]</a>.</p> <p>SAOS-2 cells were transfected with Gal4-Luc and Gal4-E2F1 plasmids in the presence or absence of RB or RB-K.</p> <p>A plasmid expressing Renilla luciferase from the thymidine kinase promoter was included in the transfections for normalization of transfection efficiency.</p> <p>The firefly and Renilla luciferase activities were measured in each sample, the ratio of which was compared among the different transfections with that from the Gal4-Luc only transfected sample as the baseline.</p> <p>The values shown are averages and standard errors from three independent experiments.</p></div

A) Transcription repression.

<div><p>The <i>Cyclin A-Luc</i> reporter plasmid contains a firefly luciferase expression cassette preceded with a fragment from the human <i>Cyclin A</i> promoter containing E2F-binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Strobeck1" target="_blank">[24]</a>.</p> <p>The reporter was transfected into SAOS-2 cells with the indicated RB expression plasmid and the control Renilla luciferase reporter.</p> <p>The normalized firefly luciferase activity from vector co-transfected cells was set at 100%.</p> <p>B) GST pull-down assay.</p> <p>Lysates from 293T cells expressing HA-tagged E2F1 and DP1 were incubated with the indicated GST-RB and GST-E7 proteins immobilized on glutathione Sepharose.</p> <p>The HA-E2F1 in the bound fraction was resolved by SDS-PAGE and detected by immunoblotting with an anti-HA antibody (α-HA).</p> <p>C) Co-immunoprecipitation. The RB-deficient C33A cells were co-transfected with HA-E2F1, DP1 and the indicated RB expression plasmids.</p> <p>Whole cell lysates were incubated with anti-RB antibody (α-RB), and the co-immunoprecipitated E2F1 examined by immunoblotting with an anti-HA antibody (α-HA).</p> <p>D) GST-pull down assay with E2F3.</p> <p>Lysates from 293T cells expressing HA-tagged E2F3 and DP1 were incubated with the indicated GST-RB and GST-E7 proteins immobilized on glutathione Separose.</p> <p>The amount of E2F3 in the bound fraction was determined by immunoblotting with anti-HA antibody (α-HA).</p></div