scholarly journals PAK1 Regulates MEC-17 Acetyltransferase Activity and Microtubule Acetylation during Proplatelet Extension

2020 ◽  
Vol 21 (20) ◽  
pp. 7531
Author(s):  
Juliette van Dijk ◽  
Guillaume Bompard ◽  
Gabriel Rabeharivelo ◽  
Julien Cau ◽  
Claude Delsert ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Cell Model ◽  
Blood Stream ◽  
Downstream Target ◽  
Platelet Release ◽  
Proplatelet Formation ◽  
P21 Activated Kinase ◽  
Previous Demonstration ◽  
Direct Regulator ◽  
Platelet Formation

Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.

In Vitro Production Of Megakaryocytes and Platelets From Human Induced Pluripotent Cells By GMP Compatible Methods

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2401-2401 ◽  
Author(s):  
Thomas Moreau ◽  
Maria Colzani ◽  
Meera Arumugam ◽  
Amanda Evans ◽  
Marloes Renee Tijssen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Transmembrane Proteins ◽  
In Vitro Production ◽  
Platelet Release ◽  
Proplatelet Formation ◽  
Induced Pluripotent ◽  
Vitro Production ◽  
Platelet Formation

Abstract Human pluripotent stem cells and in particular induced pluripotent stem cells (iPSC) derived from adult tissue have recently opened novel and promising avenues for cell therapy. In vitro production of high demand transfusion products such as platelets from HLA- and HPA-typed iPSC lines is an attractive target but to achieve this potential there is a clear need for reliable and efficient GMP compliant differentiation protocols and dedicated cell culture devices compatible with large scale cell production. We first developed a novel protocol for production of megakaryocytes (MKs) from human iPSCs based on the ectopic expression of specific transcription factors (TFs), so called “forward programming”. Through a transcriptome and protein interactome guided process, we initially shortlisted 46 TF candidates and eventually tested fourteen of the top ranked factors for their forward programming potential. Using lentiviral vectors we identified a minimal combination of three TFs which generated the highest amount of MKs. This forward programming protocol was able to robustly induce MK differentiation from various embryonic and iPSC lines. Further developments have lead to a fully standardise differentiation system using forced aggregation of defined amounts of single iPSCs into embryoid bodies and chemically defined media throughout the culture. We reliably achieve a 99% pure MK culture with a mature phenotype (i.e. CD41a+/CD61+/CD42a+/CD42b+/GP6+, polyploid and forming proplatelets and expressing key regulatory genes (e.g. NFE2, MEIS1, PBX1, RUNX1, ZFPM1) and a cell yield in excess of 50 times the initial iPSC input. These results surpass existing protocols offering wider perspectives in using human iPSC derived MKs for biological studies as well as clinical applications. This field of research has also been hampered by the lack of efficient, GMP-compatible protocols to derive platelets from MKs. We set out to address this issue by creating a 3-dimensional biomimetic/biocompatible scaffold to be incorporated a perfusion bioreactor to mimic the chemico-mechanical signals to enhance proplatelet formation and platelet release from MKs and allow the harvest of functional platelets in numbers compatible with those required for human use. The importance of the niche environment (including cell-to-cell contact and extracellular matrix proteins) for both MK maturation and platelet release has been demonstrated in different studies. Based on protein expression data from endothelial cells and mesenchymal cell lines that support platelet formation in co-cultures, we have generated a library of recombinant transmembrane proteins (TMPs). These TMPs contain the extracellular domain of the candidate transmembrane proteins at the N-terminal while at the C-terminus they contain a sequence which allows purification and immobilization on either culture plates or 3D scaffolds. We have used a combinatorial approach to perform a high throughput screening of the TMP library identify which TMPs promote proplatelet formation. Briefly, TMPs were immobilized onto 96-well plates onto which mature culture-derived MKs were seeded and proplatelet formation recorded by microscopy and digital imaging analysis. The best candidate combinations were identified through a mathematical model adapted from drug screening experiments. Finally we have produced porous matrices using GMP-grade collagen and fibrinogen via a freeze-drying process whereby ice crystals introduced into a protein-acid slurry are sublimated off to produce a fully interconnected porous structure. The structure can be tailored in terms of pore sizes and morphologies by varying the temperature profile. The structural integrity over prolonged periods in tissue culture can be enhanced by chemical crosslinking, which also removes the active moiety of collagen. We show that both collagen and fibrinogen scaffolds support MK culture from cord-blood derived CD34+ stem cells with no toxic effects and similar differentiation properties as with standard 2D cultures. We also show that the scaffold support platelet formation from seeded mature MKs. These inert scaffolds can be further functionalized by affixing the candidate TMPs identified above via a UV-light driven process onto the inert base scaffold to give a direct contact signal to the MKs to produce platelets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PAK and other Rho-associated kinases – effectors with surprisingly diverse mechanisms of regulation

2005 ◽  
Vol 386 (2) ◽  
pp. 201-214 ◽  
Author(s):  
Zhou-shen ZHAO ◽  
Ed MANSER
Keyword(s):  
Rho Gtpases ◽  
Cell Biology ◽  
Rho Gtpase ◽  
Rho Kinase ◽  
Molecular Switches ◽  
Eukaryotic Cell ◽  
Effector Proteins ◽  
Downstream Effector ◽  
P21 Activated Kinase ◽  
Do So

The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase–kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.

scholarly journals si-SNHG5-FOXF2 inhibits TGF-β1-induced fibrosis in human primary endometrial stromal cells by the Wnt/β-catenin signalling pathway

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Limin Liu ◽  
Guobin Chen ◽  
Taoliang Chen ◽  
Wenjuan Shi ◽  
Haiyan Hu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stromal Cells ◽  
Target Genes ◽  
Cell Model ◽  
Ex Vivo ◽  
Signalling Pathway ◽  
Endometrial Stromal Cells ◽  
Downstream Target ◽  
Related Proteins ◽  
Tgf Β1

Abstract Background Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. Methods FOXF2, TGF-β1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-β1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. Results FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-β1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-β1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/β-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/β-catenin pathway, thereby altering TGF-β1-mediated ECM aggregation in endometrial stromal cells ex vivo. Conclusions Regulation of the Wnt/β-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-β1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-β1-mediated fibrosis in primary HESCs.

scholarly journals Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions

2004 ◽  
Vol 15 (6) ◽  
pp. 2965-2977 ◽  
Author(s):  
Mary R. Stofega ◽  
Luraynne C. Sanders ◽  
Elisabeth M. Gardiner ◽  
Gary M. Bokoch
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Rho Gtpases ◽  
Breast Cancer Cells ◽  
Kinase Activity ◽  
Focal Adhesions ◽  
Breast Cancer Cell Lines ◽  
Cytoskeletal Remodeling ◽  
P21 Activated Kinase ◽  
Pak Kinase

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK–PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK–PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.

scholarly journals Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

2006 ◽  
Vol 172 (5) ◽  
pp. 759-769 ◽  
Author(s):  
Jean Paul ten Klooster ◽  
Zahara M. Jaffer ◽  
Jonathan Chernoff ◽  
Peter L. Hordijk
Keyword(s):  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Exchange Factor ◽  
Downstream Signaling ◽  
Intracellular Targeting ◽  
Dynamics And Control ◽  
P21 Activated Kinase ◽  
Gtpase Activation

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix.

scholarly journals Disrupted filamin A/αIIbβ3 interaction induces macrothrombocytopenia by increasing RhoA activity

Blood ◽  
2019 ◽  
Vol 133 (16) ◽  
pp. 1778-1788 ◽  
Author(s):  
Alessandro Donada ◽  
Nathalie Balayn ◽  
Dominika Sliwa ◽  
Larissa Lordier ◽  
Valentina Ceglia ◽  
...  
Keyword(s):  
Cell Model ◽  
Filamin A ◽  
Rhoa Activity ◽  
Cellular Processes ◽  
Rhoa Activation ◽  
Actomyosin Contractility ◽  
Stem Cell Model ◽  
Proplatelet Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αIIbβ3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbβ3-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αIIbβ3 interaction.

scholarly journals Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization

1998 ◽  
Vol 18 (1) ◽  
pp. 130-140 ◽  
Author(s):  
Thomas Leung ◽  
Xiang-Qun Chen ◽  
Ivan Tan ◽  
Edward Manser ◽  
Louis Lim
Keyword(s):  
Myotonic Dystrophy ◽  
Rho Gtpases ◽  
Pleckstrin Homology Domain ◽  
Cell Periphery ◽  
Cytoskeletal Reorganization ◽  
Binding Domains ◽  
Downstream Effector ◽  
Kinase C ◽  
Multidomain Structure ◽  
P21 Activated Kinase

ABSTRACT The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ∼190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C andn-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.

Distinct localizations and roles of non-muscle myosin II during proplatelet formation and platelet release

2015 ◽  
Vol 13 (5) ◽  
pp. 851-859 ◽  
Author(s):  
I. Badirou ◽  
J. Pan ◽  
S. Souquere ◽  
C. Legrand ◽  
G. Pierron ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Platelet Release ◽  
Proplatelet Formation ◽  
Muscle Myosin

scholarly journals Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function

Blood ◽  
2015 ◽  
Vol 125 (12) ◽  
pp. 1957-1967 ◽  
Author(s):  
Marjolein Meinders ◽  
Divine I. Kulu ◽  
Harmen J. G. van de Werken ◽  
Mark Hoogenboezem ◽  
Hans Janssen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Platelet Aggregation ◽  
Knockout Mice ◽  
Platelet Dysfunction ◽  
Double Knockout ◽  
Key Points ◽  
Proplatelet Formation ◽  
And Function ◽  
Platelet Formation

Key Points Megakaryocyte-specific Sp1/Sp3 double-knockout mice display thrombocytopenia, platelet dysfunction, and defects in megakaryocyte maturation. Selective Mylk inhibition by ML7 affects proplatelet formation and stabilization and ITAM receptor–mediated platelet aggregation.

Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling

2014 ◽  
Vol 307 (11) ◽  
pp. L877-L887 ◽  
Author(s):  
Jena Fediuk ◽  
Anurag S. Sikarwar ◽  
Nora Nolette ◽  
Shyamala Dakshinamurti
Keyword(s):  
Rho Gtpases ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Filamentous Actin ◽  
Actin Isoforms ◽  
Pi3k Activity ◽  
Laser Scanning Cytometry ◽  
Pulmonary Arterial ◽  
P21 Activated Kinase ◽  
Syndrome Protein

In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.

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