scholarly journals Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.

1996 ◽  
Vol 135 (6) ◽  
pp. 1515-1523 ◽  
Author(s):  
W Rodgers ◽  
J K Rose
Keyword(s):  
Kinase Activity ◽  
Specific Activity ◽  
Peptide Mapping ◽  
Tyrosine Phosphatase ◽  
T Cell Development ◽  
Jurkat Cells ◽  
Membrane Domains ◽  
Membrane Bound ◽  
Triton X ◽  
Specific Membrane

p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis

1993 ◽  
Vol 13 (3) ◽  
pp. 1464-1470
Author(s):  
S Bagrodia ◽  
S J Taylor ◽  
D Shalloway
Keyword(s):  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Src Kinase ◽  
Indirect Evidence ◽  
Membrane Localization ◽  
Wild Type ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Membrane Bound ◽  
Type C

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

scholarly journals Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

1993 ◽  
Vol 13 (3) ◽  
pp. 1464-1470 ◽  
Author(s):  
S Bagrodia ◽  
S J Taylor ◽  
D Shalloway
Keyword(s):  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Src Kinase ◽  
Indirect Evidence ◽  
Membrane Localization ◽  
Wild Type ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Membrane Bound ◽  
Type C

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

scholarly journals Palmitoylation of the three isoforms of human endothelin-converting enzyme-1

1999 ◽  
Vol 340 (3) ◽  
pp. 649-656 ◽  
Author(s):  
Anja SCHWEIZER ◽  
Bernd-Michael LÖFFLER ◽  
Jack ROHRER
Keyword(s):  
Covalent Attachment ◽  
Membrane Domains ◽  
Converting Enzyme ◽  
Wild Type ◽  
Immunofluorescence Analysis ◽  
Sds Page ◽  
Endothelin Converting Enzyme ◽  
Membrane Bound ◽  
Triton X

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation.

scholarly journals Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets.

1993 ◽  
Vol 13 (3) ◽  
pp. 1863-1871 ◽  
Author(s):  
E A Clark ◽  
J S Brugge
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Phosphatase ◽  
Specific Activity ◽  
Tyrosine Phosphatase ◽  
Insoluble Fraction ◽  
Integrin Receptor ◽  
Triton X ◽  
Autophosphorylation Site ◽  
Stimulation Of ◽  
Sustained Increase

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.

Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets

1993 ◽  
Vol 13 (3) ◽  
pp. 1863-1871
Author(s):  
E A Clark ◽  
J S Brugge
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Phosphatase ◽  
Specific Activity ◽  
Tyrosine Phosphatase ◽  
Insoluble Fraction ◽  
Integrin Receptor ◽  
Triton X ◽  
Autophosphorylation Site ◽  
Stimulation Of ◽  
Sustained Increase

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.

scholarly journals The CD20 Calcium Channel Is Localized to Microvilli and Constitutively Associated with Membrane Rafts

2004 ◽  
Vol 279 (19) ◽  
pp. 19893-19901 ◽  
Author(s):  
Haidong Li ◽  
Linda M. Ayer ◽  
Maria J. Polyak ◽  
Cathlin M. Mutch ◽  
Ryan J. Petrie ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Kinase Activity ◽  
Biological Effects ◽  
Membrane Rafts ◽  
Cross Linking ◽  
Store Operated Calcium Entry ◽  
Detergent Resistant Membranes ◽  
Punctate Pattern ◽  
Triton X ◽  
Specific Membrane

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.

scholarly journals The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 218
Author(s):  
Maria N. Balatskaya ◽  
Alexandra I. Baglay ◽  
Alexander V. Balatskiy
Keyword(s):  
Positive Control ◽  
Human Platelets ◽  
Membrane Domains ◽  
Chain Reaction ◽  
Post Translational Modifications ◽  
Reaction Data ◽  
Polymerase Chain ◽  
Triton X ◽  
Specific Membrane ◽  
Polymerase Chain Reaction Data

The membrane of platelets contains at least one uncharacterized glycosylphosphatidylinositol (GPI)-anchored protein according to the literature. Moreover, there is not enough knowledge on the receptor of low-density lipoproteins (LDL) mediating rapid Ca2+ signaling in platelets. Coincidentally, expression of a GPI-anchored protein T-cadherin increases LDL-induced Ca2+ signaling in nucleated cells. Here we showed evidence that supports the hypothesis about the presence of T-cadherin on platelets. The presence of T-cadherin on the surface of platelets and megakaryocytes was proven using antibodies whose specificity was tested on several negative and positive control cells by flow cytometry and confocal microscopy. Using phosphatidylinositol-specific phospholipase C, the presence of glycosylphosphatidylinositol anchor in the platelet T-cadherin form as well as in other known forms was confirmed. We showed by immunoblotting that the significant part of T-cadherin was detected in specific membrane domains (detergent Triton X-114 resistant) and the molecular weight of this newly identified protein was greater than that of T-cadherin from nucleated cells. Nevertheless, polymerase chain reaction data confirmed only the presence of isoform-1 of T-cadherin in platelets and megakaryocytes, which was also present in nucleated cells. We observed the redistribution of this newly identified protein after the activation of platelets, but only further work may explain its functional importance. Thus, our data described T-cadherin with some post-translational modifications as a new GPI-anchored protein on human platelets.

scholarly journals Tobacco mosaic virus for the targeted delivery of drugs to cells expressing prostate-specific membrane antigen

RSC Advances ◽  
2021 ◽  
Vol 11 (33) ◽  
pp. 20101-20108
Author(s):  
Sourabh Shukla ◽  
Isaac Marks ◽  
Derek Church ◽  
Soo-Khim Chan ◽  
Jonathan K. Pokorski ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Targeted Delivery ◽  
Prostate Gland ◽  
Prostate Specific Membrane Antigen ◽  
Important Target ◽  
Membrane Bound ◽  
Prostate Cancers ◽  
Specific Membrane

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document