Subhashree Pradhan

Overexpression of Separase induces aneuploidy and mammary tumorigenesis

Separase is an endopeptidase that separates sister chromatids by cleaving cohesin Rad21 during the metaphase-to-anaphase transition. Conditional expression of Separase in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells with both p53 WT and mutant (Ser-233-234) alleles of unknown physiological significance develops aneuploidy within 5 days of Separase induction in vitro. Overexpression of Separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In an in vivo mouse mammary transplant model, induction of Separase expression in the transplanted FSK3 cells for 3–4 weeks results in the formation of aneuploid tumors in the mammary gland. Xenograft studies combined with histological and cytogenetic analysis reveal that Separase-induced tumors are clonal in their genomic complements and have a mesenchymal phenotype suggestive of an epithelial–mesenchymal transition. Induction of Separase resulted in trisomies for chromosomes 8, 15, and 17; monosomy for chromosome 10; and amplification of the distal region of chromosomes 8 and 11. Separase protein is found to be significantly overexpressed in human breast tumors compared with matched normal tissue. These results collectively suggest that Separase is an oncogene, whose overexpression alone in mammary epithelial cells is sufficient to induce aneuploidy and tumorigenesis in a p53 mutant background.

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway.

Association between cell-derived microparticles and adverse events in patients with nonpulsatile left ventricular assist devices

BACKGROUND Continuous-flow left ventricular assist devices (LVADs) expose blood cells to high shear stress, potentially resulting in the production of microparticles that express phosphatidylserine (PS+) and promote coagulation and inflammation. In this prospective study, we attempted to determine whether PS+ microparticle levels correlate with clinical outcomes in LVAD-supported patients. METHODS We enrolled 20 patients undergoing implantation of the HeartMate II LVAD (Thoratec Corp, Pleasanton, CA) and 10 healthy controls who provided reference values for the microparticle assays. Plasma was collected before LVAD implantation, at discharge, at the 3-month follow-up, and when an adverse clinical event occurred. We quantified PS+ microparticles in the plasma using flow cytometry. RESULTS During the study period, 8 patients developed adverse clinical events: ventricular tachycardia storm in 1, non-ST-elevation myocardial infarction in 2, arterial thrombosis in 2, gastrointestinal bleeding in 2, and stroke in 3. Levels of PS+ microparticles were higher in patients at baseline than in healthy controls (2.11% ± 1.26% vs 0.69% ± 0.46%, p = 0.007). After LVAD implantation, patient PS+ microparticle levels increased to 2.39% ± 1.22% at discharge and then leveled to 1.97% ± 1.25% at the 3-month follow-up. Importantly, levels of PS+ microparticles were significantly higher in patients who developed an adverse event than in patients with no events (3.82% ± 1.17% vs 1.57% ± 0.59%, p < 0.001), even though the 2 patient groups did not markedly differ in other clinical and hematologic parameters. CONCLUSIONS Our results suggest that an elevation of PS+ microparticle levels may be associated with adverse clinical events. Thus, measuring PS+ microparticle levels in LVAD-supported patients may help identify patients at increased risk for adverse events.

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells

Background: Secretion of Weibel–Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. Objective and method: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. Results: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell‐permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell‐stimulating agent. PP2B inhibitor‐induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA‐treated HUVECs, suggesting that this process may be involved in CsA‐mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA‐treated mice, and small interfering RNA‐mediated knockdown of the α and β isoforms of the PP2B‐A subunit in HUVECs enhanced VWF secretion. Conclusions: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

Background Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear. Methodology/Principal Findings Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets. Conclusions/Significance These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.

Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

Integrin αIIbβ3 signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing αIIbβ3 to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 αIIbβ3 cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 αIIbβ3 cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 αIIbβ3 cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 αIIbβ3 cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate αIIbβ3 adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune αIIbβ3 outside-in signaling.

Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirins ability to irreversibly inhibit COX-1–mediated platelet activation, thereby blocking platelet–cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial–mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, β-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirins chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142–52. ©2016 AACR.

The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside-in αII b β3 integrin signaling.

Calcium‐dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium‐activated protease and kinase, the contribution of calcium‐activated protein serine/threonine phosphatase in platelet activation is poorly understood.

Lipid rafts contribute to agonist‐induced serine/threonine phosphatase activation and platelet aggregation

Lipid rafts are distinct microdomains in the plasma membrane with highly ordered structure due to the presence of cholesterol and sphingolipids. Following receptor engagement, certain adaptor and signaling proteins translocate in and out of lipid rafts to facilitate signaling [1]. Lipid rafts play a crucial role in platelet function by hosting a number of agonist receptors and signaling molecules [2-6]. Despite the appreciation that signaling events in rafts contribute to platelet function, whether serine/threonine (Ser/Thr) phosphatases reside in platelet rafts remains unexplored. Here, we present evidence for a partial translocation of the catalytic subunits of protein phosphatase 1 (PP1c; ppp1c) and protein phosphatase 2A (PP2Ac; ppp2Ac) into the lipid rafts in thrombin and collagen-related peptide (CRP) stimulated platelets, an event that participates in phosphatase activation and platelet aggregation. Isolation of rafts was achieved by sucrose density gradient centrifugation of 1% Triton X-100 platelet lysate followed by twelve equal fractionations from the top of the gradient [4]. Dot blot staining of all the fractions with cholera toxin B-subunit, which specifically binds to lipid raft marker ganglioside GM1, revealed that lipid rafts predominantly localized to fractions 2-4 with a minor presence in fraction 5 (Figure 1A, first panel). Immunoblotting of the fractions from resting platelets with anti-PP1c or anti-PP2Ac antibodies revealed the presence of PP1c (Figure 1A, second panel) and PP2Ac (Figure 1B, first panel) in the late raft fractions 4 and 5. However, the majority of PP1c/PP2Ac was localized to the non-lipid raft fractions (6 through 12). Following platelet activation with either thrombin or CRP, additional PP1c (Figure 1A, third and sixth panel) and PP2Ac (Figure 1B, second and fifth panel) were prominently detected in the early raft fractions 2 and 3 (thrombin) and fraction 3 (CRP). These observations indicate that the engagement of protease activated receptor 1 (PAR1) and the GPVI-FcRγ receptor in platelets is associated with the partial translocation of Ser/Thr phosphatases to the lipid rafts. Consistent with these observations, the receptors for thrombin (PAR1) [7] and CRP (GPVI) [5] also partition to platelet lipid rafts. Ser/Thr phosphatases facilitated PAR1 and GPVI mediated platelet aggregation because compared to the control DMSO, low dose inhibitors of PP1/PP2A (tautomycin) or PP2A (okadaic acid) revealed a modest but significant reduction in thrombin (Figures 1C) and CRP (Figures 1D) induced aggregation. These observations are consistent with the previous findings that platelet functions are impaired by pharmacological inhibitors that block the activity of Ser/Thr phosphatases [8-10]. Figure 1 Washed platelets (resting or stimulated with 0.5 U/ml thrombin, 5 μg/ml CRP for 2 minutes) were lysed using ice cold 1% Triton X-100 lysis buffer. In some experiments, platelets were incubated with 10 mM MβCD or 10 mM αCD for 30 ... Disruption of platelet rafts by methyl-β-cyclodextrin (MβCD), which depletes membrane cholesterol prior to thrombin or CRP treatment, resulted in the loss of PP1c (Figure 1A, fourth and seventh panel) and PP2Ac (Figure 1B, third and sixth panel) from the early raft fractions 2 and 3. Since MβCD may exhibit raft dependent and independent effects [11], α cyclodextrin (αCD), an inactive cyclodextrin analogue was utilized as a control to demonstrate raft specificity [12]. PP1c (Figure 1A, fifth and eighth panel) and PP2Ac (Figures 1B, fourth and seventh panel) were retained in the lipid rafts when agonist-stimulated platelets were pretreated with αCD. How PP1c/PP2Ac localizes to the lipid rafts is unclear. PP1c and PP2Ac exhibit several cytosine residues in close proximity and may undergo palmitoylation, a modification that facilitates raft localization. In 3T3 cells, PP2A was localized to lipid rafts via its association with the cholesterol-regulated scaffolding protein OSBP [13]. To evaluate if the localization of phosphatases to rafts following agonist stimulation affected its activity, we disrupted rafts and evaluated phosphatase activity. Compared to the resting platelets, treatment with thrombin and CRP resulted in a moderate but significant increase in PP1c (Figure 1E) and PP2Ac (Figure 1F) activity. PP1c and PP2Ac enzymatic activities were specific because the mouse IgG immunoprecipitates detected only the base line phosphate levels (~100-150 pmoles of phosphate/minute) (not shown). Raft disruption by MβCD, but not by αCD decreased agonist-induced activation of PP1c and PP2Ac (Figures 1E and 1F). The amount of phosphatases available for the activity assays in the immune precipitates was comparable across various treatments (Figure 1G). Furthermore, agonist-induced platelet aggregation was also impaired in MβCD but not in αCD treated platelets (Figures 1C and 1D). Thus, disrupting lipid rafts reduced agonist-induced phosphatase activation with a concomitant impairment in platelet aggregation. To investigate if raft localization of phosphatases influenced platelet function, we assessed the impact of PP1c/PP2Ac inhibitors on platelet aggregation in the presence of a raft disruptor. Compared to platelets treated with only Ser/Thr phosphatase inhibitors, a combination of Ser/Thr phosphatase inhibitors and MβCD treatment significantly decreased agonist-induced platelet aggregation (Figures 1C and 1D). This suggests that the association of Ser/Thr phosphatases with rafts contribute to platelet aggregation. MβCD treatment does not alter integrin αIIbβ3 surface expression [4] and cannot account for the decreased aggregation. To summarize, previous studies have identified kinases (Syk, palmitolyated Fyn and Lyn) but not phosphatases in rafts. We show that Ser/Thr phosphatases can localize to lipid rafts following platelet activation with thrombin and CRP. Translocation of Ser/Thr phosphatases to lipid rafts facilitates complete agonist-induced phosphatase activation and platelet aggregation.

A novel interaction of the catalytic subunit of protein phosphatase 2A with the adaptor protein CIN85 suppresses phosphatase activity and facilitates platelet outside-in αIIbβ3 integrin signaling

The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block (396PAIPPKKPRP405) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395–407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.