hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation

Background: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store‐operated Ca2+ entry (SOCE). Objectives: To assess hTRPC phosphotyrosine content upon platelet stimulation. Met...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Journal of thrombosis and haemostasis 2007-12, Vol.5 (12), p.2476-2483
Hauptverfasser: REDONDO, P. C., HARPER, A. G. S., HARPER, M. T., BROWNLOW, S. L., ROSADO, J. A., SAGE, S. O.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 2483
container_issue 12
container_start_page 2476
container_title Journal of thrombosis and haemostasis
container_volume 5
creator REDONDO, P. C.
HARPER, A. G. S.
HARPER, M. T.
BROWNLOW, S. L.
ROSADO, J. A.
SAGE, S. O.
description Background: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store‐operated Ca2+ entry (SOCE). Objectives: To assess hTRPC phosphotyrosine content upon platelet stimulation. Methods: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. Results: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of α‐actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr‐evoked α‐actinin tyrosine phosphorylation was increased by inhibiting the α‐actinin phosphatase, SHP‐1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca2+ sensor STIM1. Conclusions: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.
doi_str_mv 10.1111/j.1538-7836.2007.02773.x
format Article
fullrecord <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_68537948</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>68537948</sourcerecordid><originalsourceid>FETCH-LOGICAL-c4173-bd0e49b33bdb1453086589324056da20ed41f2d5a1122030eef86ab8c308a66a3</originalsourceid><addsrcrecordid>eNqNkEtOwzAURS0EolDYAvKIURv8yccZMEAVUFAlECpjy4kd6ipxSuxAM2MJbIWNsAhWQtIUmGLJ8rN93rV8AIAYebgdZ0sPB5SNI0ZDjyAUeYhEEfXWO-Dg92L3p44pHYBDa5cI4TggaB8McMRiElB8ANxi_nA_wV9v78LaMtXCKQk_P7p96rTRZgSFkdCUDvYk1M6qPBtBbaFrqtJqo-BqUdp2Vk2-6Zd1pc0TXNSFMHDVneXKwS7wRThdmiOwl4ncquPtOgSPV5fzyXQ8u7u-mVzMxqmPIzpOJFJ-nFCayAT7AUUsDFhMiY-CUAqClPRxRmQgMCYEUaRUxkKRsLQlRRgKOgSnfe6qKp9rZR0vtE1VngujytrykAU0in3WgqwH0_ZDtlIZX1W6EFXDMeKdcb7knUzeieWdcb4xztdt68n2jToplPxr3CpugfMeeNW5av4dzG_n066i34utkpw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>68537948</pqid></control><display><type>article</type><title>hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation</title><source>MEDLINE</source><source>EZB-FREE-00999 freely available EZB journals</source><source>Alma/SFX Local Collection</source><creator>REDONDO, P. C. ; HARPER, A. G. S. ; HARPER, M. T. ; BROWNLOW, S. L. ; ROSADO, J. A. ; SAGE, S. O.</creator><creatorcontrib>REDONDO, P. C. ; HARPER, A. G. S. ; HARPER, M. T. ; BROWNLOW, S. L. ; ROSADO, J. A. ; SAGE, S. O.</creatorcontrib><description>Background: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store‐operated Ca2+ entry (SOCE). Objectives: To assess hTRPC phosphotyrosine content upon platelet stimulation. Methods: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. Results: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of α‐actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr‐evoked α‐actinin tyrosine phosphorylation was increased by inhibiting the α‐actinin phosphatase, SHP‐1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca2+ sensor STIM1. Conclusions: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.</description><identifier>ISSN: 1538-7933</identifier><identifier>ISSN: 1538-7836</identifier><identifier>EISSN: 1538-7836</identifier><identifier>DOI: 10.1111/j.1538-7836.2007.02773.x</identifier><identifier>PMID: 17892531</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Actinin - metabolism ; Blood Platelets - drug effects ; Blood Platelets - enzymology ; Blood Platelets - metabolism ; Blotting, Western ; Calcium Signaling - drug effects ; Enzyme Inhibitors - pharmacology ; Humans ; Immunoprecipitation - methods ; In Vitro Techniques ; Membrane Proteins - metabolism ; Multiprotein Complexes - metabolism ; Neoplasm Proteins - metabolism ; Phosphorylation ; Phosphotyrosine - metabolism ; platelet ; Platelet Activation - drug effects ; ProCISA ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 - antagonists &amp; inhibitors ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 - metabolism ; Protein-Tyrosine Kinases - metabolism ; Quinolines - pharmacology ; STIM1 ; store‐operated Ca2+ entry ; Stromal Interaction Molecule 1 ; Thrombin - metabolism ; Time Factors ; transient receptor potential channels ; TRPC Cation Channels - metabolism ; Tyrosine - metabolism ; tyrosine phosphorylation ; α‐actinin</subject><ispartof>Journal of thrombosis and haemostasis, 2007-12, Vol.5 (12), p.2476-2483</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4173-bd0e49b33bdb1453086589324056da20ed41f2d5a1122030eef86ab8c308a66a3</citedby><cites>FETCH-LOGICAL-c4173-bd0e49b33bdb1453086589324056da20ed41f2d5a1122030eef86ab8c308a66a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17892531$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>REDONDO, P. C.</creatorcontrib><creatorcontrib>HARPER, A. G. S.</creatorcontrib><creatorcontrib>HARPER, M. T.</creatorcontrib><creatorcontrib>BROWNLOW, S. L.</creatorcontrib><creatorcontrib>ROSADO, J. A.</creatorcontrib><creatorcontrib>SAGE, S. O.</creatorcontrib><title>hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation</title><title>Journal of thrombosis and haemostasis</title><addtitle>J Thromb Haemost</addtitle><description>Background: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store‐operated Ca2+ entry (SOCE). Objectives: To assess hTRPC phosphotyrosine content upon platelet stimulation. Methods: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. Results: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of α‐actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr‐evoked α‐actinin tyrosine phosphorylation was increased by inhibiting the α‐actinin phosphatase, SHP‐1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca2+ sensor STIM1. Conclusions: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.</description><subject>Actinin - metabolism</subject><subject>Blood Platelets - drug effects</subject><subject>Blood Platelets - enzymology</subject><subject>Blood Platelets - metabolism</subject><subject>Blotting, Western</subject><subject>Calcium Signaling - drug effects</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Humans</subject><subject>Immunoprecipitation - methods</subject><subject>In Vitro Techniques</subject><subject>Membrane Proteins - metabolism</subject><subject>Multiprotein Complexes - metabolism</subject><subject>Neoplasm Proteins - metabolism</subject><subject>Phosphorylation</subject><subject>Phosphotyrosine - metabolism</subject><subject>platelet</subject><subject>Platelet Activation - drug effects</subject><subject>ProCISA</subject><subject>Protein Tyrosine Phosphatase, Non-Receptor Type 6 - antagonists &amp; inhibitors</subject><subject>Protein Tyrosine Phosphatase, Non-Receptor Type 6 - metabolism</subject><subject>Protein-Tyrosine Kinases - metabolism</subject><subject>Quinolines - pharmacology</subject><subject>STIM1</subject><subject>store‐operated Ca2+ entry</subject><subject>Stromal Interaction Molecule 1</subject><subject>Thrombin - metabolism</subject><subject>Time Factors</subject><subject>transient receptor potential channels</subject><subject>TRPC Cation Channels - metabolism</subject><subject>Tyrosine - metabolism</subject><subject>tyrosine phosphorylation</subject><subject>α‐actinin</subject><issn>1538-7933</issn><issn>1538-7836</issn><issn>1538-7836</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkEtOwzAURS0EolDYAvKIURv8yccZMEAVUFAlECpjy4kd6ipxSuxAM2MJbIWNsAhWQtIUmGLJ8rN93rV8AIAYebgdZ0sPB5SNI0ZDjyAUeYhEEfXWO-Dg92L3p44pHYBDa5cI4TggaB8McMRiElB8ANxi_nA_wV9v78LaMtXCKQk_P7p96rTRZgSFkdCUDvYk1M6qPBtBbaFrqtJqo-BqUdp2Vk2-6Zd1pc0TXNSFMHDVneXKwS7wRThdmiOwl4ncquPtOgSPV5fzyXQ8u7u-mVzMxqmPIzpOJFJ-nFCayAT7AUUsDFhMiY-CUAqClPRxRmQgMCYEUaRUxkKRsLQlRRgKOgSnfe6qKp9rZR0vtE1VngujytrykAU0in3WgqwH0_ZDtlIZX1W6EFXDMeKdcb7knUzeieWdcb4xztdt68n2jToplPxr3CpugfMeeNW5av4dzG_n066i34utkpw</recordid><startdate>200712</startdate><enddate>200712</enddate><creator>REDONDO, P. C.</creator><creator>HARPER, A. G. S.</creator><creator>HARPER, M. T.</creator><creator>BROWNLOW, S. L.</creator><creator>ROSADO, J. A.</creator><creator>SAGE, S. O.</creator><general>Blackwell Publishing Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200712</creationdate><title>hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation</title><author>REDONDO, P. C. ; HARPER, A. G. S. ; HARPER, M. T. ; BROWNLOW, S. L. ; ROSADO, J. A. ; SAGE, S. O.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4173-bd0e49b33bdb1453086589324056da20ed41f2d5a1122030eef86ab8c308a66a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Actinin - metabolism</topic><topic>Blood Platelets - drug effects</topic><topic>Blood Platelets - enzymology</topic><topic>Blood Platelets - metabolism</topic><topic>Blotting, Western</topic><topic>Calcium Signaling - drug effects</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Humans</topic><topic>Immunoprecipitation - methods</topic><topic>In Vitro Techniques</topic><topic>Membrane Proteins - metabolism</topic><topic>Multiprotein Complexes - metabolism</topic><topic>Neoplasm Proteins - metabolism</topic><topic>Phosphorylation</topic><topic>Phosphotyrosine - metabolism</topic><topic>platelet</topic><topic>Platelet Activation - drug effects</topic><topic>ProCISA</topic><topic>Protein Tyrosine Phosphatase, Non-Receptor Type 6 - antagonists &amp; inhibitors</topic><topic>Protein Tyrosine Phosphatase, Non-Receptor Type 6 - metabolism</topic><topic>Protein-Tyrosine Kinases - metabolism</topic><topic>Quinolines - pharmacology</topic><topic>STIM1</topic><topic>store‐operated Ca2+ entry</topic><topic>Stromal Interaction Molecule 1</topic><topic>Thrombin - metabolism</topic><topic>Time Factors</topic><topic>transient receptor potential channels</topic><topic>TRPC Cation Channels - metabolism</topic><topic>Tyrosine - metabolism</topic><topic>tyrosine phosphorylation</topic><topic>α‐actinin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>REDONDO, P. C.</creatorcontrib><creatorcontrib>HARPER, A. G. S.</creatorcontrib><creatorcontrib>HARPER, M. T.</creatorcontrib><creatorcontrib>BROWNLOW, S. L.</creatorcontrib><creatorcontrib>ROSADO, J. A.</creatorcontrib><creatorcontrib>SAGE, S. O.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of thrombosis and haemostasis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>REDONDO, P. C.</au><au>HARPER, A. G. S.</au><au>HARPER, M. T.</au><au>BROWNLOW, S. L.</au><au>ROSADO, J. A.</au><au>SAGE, S. O.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation</atitle><jtitle>Journal of thrombosis and haemostasis</jtitle><addtitle>J Thromb Haemost</addtitle><date>2007-12</date><risdate>2007</risdate><volume>5</volume><issue>12</issue><spage>2476</spage><epage>2483</epage><pages>2476-2483</pages><issn>1538-7933</issn><issn>1538-7836</issn><eissn>1538-7836</eissn><abstract>Background: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store‐operated Ca2+ entry (SOCE). Objectives: To assess hTRPC phosphotyrosine content upon platelet stimulation. Methods: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. Results: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of α‐actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr‐evoked α‐actinin tyrosine phosphorylation was increased by inhibiting the α‐actinin phosphatase, SHP‐1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca2+ sensor STIM1. Conclusions: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>17892531</pmid><doi>10.1111/j.1538-7836.2007.02773.x</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 1538-7933
ispartof Journal of thrombosis and haemostasis, 2007-12, Vol.5 (12), p.2476-2483
issn 1538-7933
1538-7836
1538-7836
language eng
recordid cdi_proquest_miscellaneous_68537948
source MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection
subjects Actinin - metabolism
Blood Platelets - drug effects
Blood Platelets - enzymology
Blood Platelets - metabolism
Blotting, Western
Calcium Signaling - drug effects
Enzyme Inhibitors - pharmacology
Humans
Immunoprecipitation - methods
In Vitro Techniques
Membrane Proteins - metabolism
Multiprotein Complexes - metabolism
Neoplasm Proteins - metabolism
Phosphorylation
Phosphotyrosine - metabolism
platelet
Platelet Activation - drug effects
ProCISA
Protein Tyrosine Phosphatase, Non-Receptor Type 6 - antagonists & inhibitors
Protein Tyrosine Phosphatase, Non-Receptor Type 6 - metabolism
Protein-Tyrosine Kinases - metabolism
Quinolines - pharmacology
STIM1
store‐operated Ca2+ entry
Stromal Interaction Molecule 1
Thrombin - metabolism
Time Factors
transient receptor potential channels
TRPC Cation Channels - metabolism
Tyrosine - metabolism
tyrosine phosphorylation
α‐actinin
title hTRPC1‐associated α‐actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T22%3A21%3A32IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=hTRPC1%E2%80%90associated%20%CE%B1%E2%80%90actinin,%20and%20not%20hTRPC1%20itself,%20is%20tyrosine%20phosphorylated%20during%20human%20platelet%20activation&rft.jtitle=Journal%20of%20thrombosis%20and%20haemostasis&rft.au=REDONDO,%20P.%20C.&rft.date=2007-12&rft.volume=5&rft.issue=12&rft.spage=2476&rft.epage=2483&rft.pages=2476-2483&rft.issn=1538-7933&rft.eissn=1538-7836&rft_id=info:doi/10.1111/j.1538-7836.2007.02773.x&rft_dat=%3Cproquest_cross%3E68537948%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=68537948&rft_id=info:pmid/17892531&rfr_iscdi=true