Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation

2001 ◽  
Vol 356 (3) ◽  
pp. 875-881 ◽  
Author(s):  
Isabelle LOPEZ ◽  
Véronique DUPREZ ◽  
Josiane MELLE ◽  
François DREYFUS ◽  
Sylviane LÉVY-TOLÉDANO ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Shape Change ◽  
Insoluble Fraction ◽  
Actin Binding ◽  
Syk Kinase ◽  
Tyrosine Phosphorylated Protein ◽  
Triton X ◽  
Cortactin Tyrosine Phosphorylation ◽  
Platelet Shape

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Platelet Aggregation Caused by Dithiothreitol

1984 ◽  
Vol 51 (01) ◽  
pp. 119-124 ◽  
Author(s):  
M B Zucker ◽  
N C Masiello
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Electrophoretic Patterns ◽  
Discernible Effect ◽  
Variable Extent ◽  
Triton X ◽  
Induced Aggregation ◽  
Platelet Shape

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

scholarly journals Recycling of platelet phosphorylation and cytoskeletal assembly.

1984 ◽  
Vol 98 (1) ◽  
pp. 8-15 ◽  
Author(s):  
A C Cox ◽  
R C Carroll ◽  
J G White ◽  
G H Rao
Keyword(s):  
Protein Phosphorylation ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Binding Protein ◽  
Actin Binding ◽  
Dibutyryl Camp ◽  
Actin Binding Protein ◽  
Fibrinogen Binding ◽  
Triton X ◽  
Subsequent Addition

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.

scholarly journals Regulation of F-Actin Binding to Platelet Moesin In Vitro by Both Phosphorylation of Threonine 558 and Polyphosphatidylinositides

1999 ◽  
Vol 10 (8) ◽  
pp. 2669-2685 ◽  
Author(s):  
Fumihiko Nakamura ◽  
Laiqiang Huang ◽  
Kersi Pestonjamasp ◽  
Elizabeth J. Luna ◽  
Heinz Furthmayr
Keyword(s):  
Kinase Inhibitor ◽  
Human Platelets ◽  
Actin Binding ◽  
Calyculin A ◽  
High K ◽  
Triton X ◽  
Cellular Control ◽  
Actin Binding Site ◽  
Nonionic Detergents

Activation of human platelets with thrombin transiently increases phosphorylation at558threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.

scholarly journals Granulocyte-macrophage colony-stimulating factor induces a staurosporine inhibitable tyrosine phosphorylation of unique neutrophil proteins

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2446-2454
Author(s):  
RL Berkow
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Triton X ◽  
Stimulating Factor

Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95- Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF- induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.

Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP

1992 ◽  
Vol 262 (3) ◽  
pp. C701-C707 ◽  
Author(s):  
A. Oda ◽  
B. J. Druker ◽  
M. Smith ◽  
E. W. Salzman
Keyword(s):  
Sodium Nitroprusside ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Shape Change ◽  
High Concentration ◽  
Serotonin Secretion ◽  
Granule Secretion ◽  
Camp And Cgmp ◽  
Platelet Shape ◽  
Platelet Functions

Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.

scholarly journals Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase

2004 ◽  
Vol 380 (2) ◽  
pp. 581-591 ◽  
Author(s):  
Lingzhi FAN ◽  
Caterina Di CIANO-OLIVEIRA ◽  
Scott A. WEED ◽  
Andrew W. B. CRAIG ◽  
Peter A. GREER ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Feedback Mechanism ◽  
Reciprocal Relationship ◽  
Actin Binding ◽  
Hypertonic Stress ◽  
Tyrosine Residues ◽  
Actin Depolymerization ◽  
N Terminus ◽  
Cortactin Tyrosine Phosphorylation

The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer–cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer–cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

scholarly journals Role of gelsolin in actin depolymerization of adherent human neutrophils.

1997 ◽  
Vol 8 (1) ◽  
pp. 121-128 ◽  
Author(s):  
J S Wang ◽  
J P Coburn ◽  
A I Tauber ◽  
K S Zaner
Keyword(s):  
Soluble Fraction ◽  
Signal Transduction Pathway ◽  
Insoluble Fraction ◽  
Actin Binding ◽  
Human Neutrophils ◽  
Nonreceptor Tyrosine Kinase ◽  
Actin Depolymerization ◽  
Herbimycin A ◽  
Triton X

Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization.

scholarly journals Cortactin Tyrosine Phosphorylation Requires Rac1 Activity and Association with the Cortical Actin Cytoskeleton

2003 ◽  
Vol 14 (8) ◽  
pp. 3216-3229 ◽  
Author(s):  
Julie A. Head ◽  
Dongyan Jiang ◽  
Min Li ◽  
Lynda J. Zorn ◽  
Erik M. Schaefer ◽  
...  
Keyword(s):  
Cell Motility ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cell Periphery ◽  
Carboxy Terminus ◽  
Cortical Actin ◽  
Cortactin Tyrosine Phosphorylation

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.

Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins

1999 ◽  
Vol 276 (5) ◽  
pp. F737-F750 ◽  
Author(s):  
Tatsuo Tsukamoto ◽  
Sanjay K. Nigam
Keyword(s):  
Tyrosine Kinase ◽  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Atp Depletion ◽  
Metabolic Inhibitors ◽  
Permeability Barrier ◽  
Density Fractions ◽  
Cell Monolayers ◽  
Triton X

After the simulation of anoxia by ATP depletion of MDCK cell monolayers with metabolic inhibitors, the tight junction (TJ) is known to become structurally perturbed, leading to loss of the permeability barrier. Peripheral TJ proteins such as zonula occludens 1 (ZO-1), ZO-2, and cingulin become extremely insoluble and associate into large macromolecular complexes (T. Tsukamoto and S. K. Nigam. J. Biol. Chem. 272: 16133–16139, 1997). For up to 3 h, this process is reversible by ATP repletion. We now show that the reassembly process depends on tyrosine phosphorylation. Recovery of transepithelial electrical resistance in ATP-replete monolayers was markedly inhibited by the tyrosine kinase inhibitor, genistein. Indirect immunofluorescence revealed a decrease in staining of occludin, a membrane component of the TJ, in the region of the TJ after ATP depletion, which reversed after ATP repletion; this reversal process was inhibited by genistein. Examination of the Triton X-100 solubilities of occludin and several nonmembrane TJ proteins revealed a shift of occludin and nonmembrane TJ proteins into an insoluble pool following ATP depletion. These changes reversed after ATP repletion, and the movement of insoluble occludin, ZO-1, and ZO-2 back into the soluble pool was again via a genistein-sensitive mechanism. Rate-zonal centrifugation analyses of detergent-soluble TJ proteins showed a reversible increase in higher density fractions following ATP depletion-repletion, although this change was not affected by genistein. In32P-labeled cells, dephosphorylation of all studied TJ proteins was observed during ATP depletion, followed by rephosphorylation during ATP repletion; rephosphorylation of occludin was inhibited by genistein. Furthermore, during the ATP repletion phase, tyrosine phosphorylation of Triton X-100-insoluble occludin, which is localized at the junction, as well as ZO-2, p130/ZO-3 (though not ZO-1), and other proteins was evident; this tyrosine phosphorylation was completely inhibited by genistein. This indicates that tyrosine kinase activity is necessary for TJ reassembly during ATP repletion and suggests an important role for the tyrosine phosphorylation of occludin, ZO-2, p130/ZO-3, and possibly other proteins in the processes involved in TJ (re)formation.

scholarly journals p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1330-1338 ◽  
Author(s):  
A Oda ◽  
K Ozaki ◽  
BJ Druker ◽  
Y Miyakawa ◽  
H Miyazaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Blood Platelets ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Genetically Engineered ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Endogenous Substrate ◽  
Tyrosine Phosphorylated Protein ◽  
Triton X

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c- Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

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