scholarly journals Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

2020 ◽  
Vol 21 (18) ◽  
pp. 6563
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Soochong Kim
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Tyrosine Kinase 2 ◽  
Platelet Spreading ◽  
Protein Kinase Cδ

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

The Role of Akt3 in Mediating Outside-in Signaling of the Platelet Integrin αIIbβ3

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1134-1134
Author(s):  
Kelly A O'Brien ◽  
Nissim Hay ◽  
Xiaoping Du
Keyword(s):  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Akt Inhibitor ◽  
Integrin Αiibβ3 ◽  
Akt Activation ◽  
Downstream Effector ◽  
Platelet Spreading ◽  
Human And Mouse

Abstract Abstract 1134 Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes

2014 ◽  
Vol 307 (12) ◽  
pp. G1207-G1215 ◽  
Author(s):  
Cynthia R. L. Webster ◽  
Andrea N. Johnston ◽  
M. Sawkat Anwer
Keyword(s):  
Protein Kinase ◽  
Rat Hepatocytes ◽  
Wild Type ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Induced Apoptosis ◽  
Hoechst Staining ◽  
Activate Protein Kinase ◽  
Protein Kinase Cδ

Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.

Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Masaru Niki ◽  
Hong Jin ◽  
Anil K Chauhan ◽  
Steven R Lentz
Keyword(s):  
Carotid Artery ◽  
Adaptor Protein ◽  
Clot Retraction ◽  
Thromboxane A2 Receptor ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Platelet Signaling ◽  
Platelet Spreading ◽  
Inside Out

Background Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined. Methods We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist U46619. Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured. Results No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P<0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P<0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P<0.05). Conclusions The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.

scholarly journals Akt isoforms differentially regulate neutrophil functions

Blood ◽  
2010 ◽  
Vol 115 (21) ◽  
pp. 4237-4246 ◽  
Author(s):  
Jia Chen ◽  
Haiyang Tang ◽  
Nissim Hay ◽  
Jingsong Xu ◽  
Richard D. Ye
Keyword(s):  
Kinase Inhibitor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Leading Edge ◽  
Neutrophil Activation ◽  
Enzyme Release ◽  
Wild Type ◽  
Akt Isoforms ◽  
Phosphoinositide 3 Kinase ◽  
O2 Production

In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O2− production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O2− production compared with wild-type and Akt1−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O2− production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O2− production in Akt2−/− neutrophils was accompanied by reduced p47phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.

scholarly journals The pathophysiologic role of the protein kinase Cδ pathway in the intervertebral discs of rabbits and mice: In vitro, ex vivo, and in vivo studies

2012 ◽  
Vol 64 (6) ◽  
pp. 1950-1959 ◽  
Author(s):  
Michael B. Ellman ◽  
Jae-Sung Kim ◽  
Howard S. An ◽  
Jeffrey S. Kroin ◽  
Xin Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ex Vivo ◽  
Intervertebral Discs ◽  
In Vivo Studies ◽  
Protein Kinase Cδ

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells

1992 ◽  
Vol 12 (7) ◽  
pp. 3305-3312
Author(s):  
M Izquierdo ◽  
J Downward ◽  
J D Graves ◽  
D A Cantrell
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Antigen Receptor ◽  
Permeabilized Cell ◽  
Regulatory Pathways ◽  
Kinase C ◽  
Stimulation Of

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Mitogen-Activated Protein Kinase Hog1 Mediates Adaptation to G1 Checkpoint Arrest during Arsenite and Hyperosmotic Stress

2008 ◽  
Vol 7 (8) ◽  
pp. 1309-1317 ◽  
Author(s):  
Iwona Migdal ◽  
Yulia Ilina ◽  
Markus J. Tamás ◽  
Robert Wysocki
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Arrest ◽  
Kinase Inhibitor ◽  
Hyperosmotic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Hog Pathway ◽  
Cycle Arrest ◽  
Mitogen Activated Protein

ABSTRACT Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1 and G2 checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G1 delay but did not cause a persistent G1 arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1 exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G1 arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1 cell cycle arrest.

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