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Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets
Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor–based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading...
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| Published in: | The Journal of biological chemistry 2023-07, Vol.299 (7), p.104865-104865, Article 104865 |
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| creator | Dangelmaier, Carol A. Patchin, Margaret Vajipayajula, Dhruv N. Vari, Hymavathi Reddy Singh, Pankaj K. Wright, Monica N. Kostyak, John C. Tsygankov, Alexander Y. Kunapuli, Satya P. |
| description | Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor–based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses. |
| doi_str_mv | 10.1016/j.jbc.2023.104865 |
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Upon phosphorylation of the platelet immunoreceptor–based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/j.jbc.2023.104865</identifier><identifier>PMID: 37268160</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Blood Platelets ; Collection: Signal Transduction ; hemostasis ; Mice ; Phosphorylation ; platelet ; Platelet Activation ; Platelet Aggregation ; Platelet Membrane Glycoproteins - genetics ; Platelet Membrane Glycoproteins - metabolism ; spleen tyrosine kinase ; Syk Kinase - genetics ; Syk Kinase - metabolism ; thrombosis ; Tyrosine</subject><ispartof>The Journal of biological chemistry, 2023-07, Vol.299 (7), p.104865-104865, Article 104865</ispartof><rights>2023 The Authors</rights><rights>Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.</rights><rights>2023 The Authors 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3675-5fd29f96e6eaea10feaf7d246edef8d0aa56178ee678ac1019425779cab25cc03</citedby><cites>FETCH-LOGICAL-c3675-5fd29f96e6eaea10feaf7d246edef8d0aa56178ee678ac1019425779cab25cc03</cites><orcidid>0000-0002-2778-2909</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10320515/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10320515/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,733,786,790,891,27957,27958,53827,53829</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37268160$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dangelmaier, Carol A.</creatorcontrib><creatorcontrib>Patchin, Margaret</creatorcontrib><creatorcontrib>Vajipayajula, Dhruv N.</creatorcontrib><creatorcontrib>Vari, Hymavathi Reddy</creatorcontrib><creatorcontrib>Singh, Pankaj K.</creatorcontrib><creatorcontrib>Wright, Monica N.</creatorcontrib><creatorcontrib>Kostyak, John C.</creatorcontrib><creatorcontrib>Tsygankov, Alexander Y.</creatorcontrib><creatorcontrib>Kunapuli, Satya P.</creatorcontrib><title>Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor–based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.</description><subject>Animals</subject><subject>Blood Platelets</subject><subject>Collection: Signal Transduction</subject><subject>hemostasis</subject><subject>Mice</subject><subject>Phosphorylation</subject><subject>platelet</subject><subject>Platelet Activation</subject><subject>Platelet Aggregation</subject><subject>Platelet Membrane Glycoproteins - genetics</subject><subject>Platelet Membrane Glycoproteins - metabolism</subject><subject>spleen tyrosine kinase</subject><subject>Syk Kinase - genetics</subject><subject>Syk Kinase - metabolism</subject><subject>thrombosis</subject><subject>Tyrosine</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><recordid>eNp9kc1u1DAUhS1ERaeFB2CDvGSTwT-x44gFqiqglVqVRZFgZXmc64yHjB3sZMS8PR6mVGWDN9aVv3N8dQ5CrylZUkLlu81ys7JLRhgvc62keIYWlChecUG_PUcLQhitWibUKTrLeUPKqVv6Ap3yhklFJVmgX1_WMY_rmPaDmXwMODqcxwEg4GmfYvYB8A8fTAZsJvyd1xIH6Au6g2GPE_Rz0UHG1_cXt9UWOl-mDmffBzP40GMTOuzmYP94-4DHAz7AlF-iE2eGDK8e7nP09dPH-8ur6ubu8_XlxU1luWxEJVzHWtdKkGDAUOLAuKZjtYQOnOqIMULSRgHIRhlbUmlrJpqmtWbFhLWEn6MPR99xXpX9LIQpmUGPyW9N2utovP73Jfi17uNOU8IZEVQUh7cPDin-nCFPeuuzhWEwAeKcNVOMcUVIywpKj6gt0eUE7vEfSvShMr3RpTJ9qEwfKyuaN08XfFT87agA748AlJh2HpLO1kOwJewEdtJd9P-x_w3h-aoy</recordid><startdate>20230701</startdate><enddate>20230701</enddate><creator>Dangelmaier, Carol A.</creator><creator>Patchin, Margaret</creator><creator>Vajipayajula, Dhruv N.</creator><creator>Vari, Hymavathi Reddy</creator><creator>Singh, Pankaj K.</creator><creator>Wright, Monica N.</creator><creator>Kostyak, John C.</creator><creator>Tsygankov, Alexander Y.</creator><creator>Kunapuli, Satya P.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><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><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-2778-2909</orcidid></search><sort><creationdate>20230701</creationdate><title>Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets</title><author>Dangelmaier, Carol A. ; Patchin, Margaret ; Vajipayajula, Dhruv N. ; Vari, Hymavathi Reddy ; Singh, Pankaj K. ; Wright, Monica N. ; Kostyak, John C. ; Tsygankov, Alexander Y. ; Kunapuli, Satya P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3675-5fd29f96e6eaea10feaf7d246edef8d0aa56178ee678ac1019425779cab25cc03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Animals</topic><topic>Blood Platelets</topic><topic>Collection: Signal Transduction</topic><topic>hemostasis</topic><topic>Mice</topic><topic>Phosphorylation</topic><topic>platelet</topic><topic>Platelet Activation</topic><topic>Platelet Aggregation</topic><topic>Platelet Membrane Glycoproteins - genetics</topic><topic>Platelet Membrane Glycoproteins - metabolism</topic><topic>spleen tyrosine kinase</topic><topic>Syk Kinase - genetics</topic><topic>Syk Kinase - metabolism</topic><topic>thrombosis</topic><topic>Tyrosine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dangelmaier, Carol A.</creatorcontrib><creatorcontrib>Patchin, Margaret</creatorcontrib><creatorcontrib>Vajipayajula, Dhruv N.</creatorcontrib><creatorcontrib>Vari, Hymavathi Reddy</creatorcontrib><creatorcontrib>Singh, Pankaj K.</creatorcontrib><creatorcontrib>Wright, Monica N.</creatorcontrib><creatorcontrib>Kostyak, John C.</creatorcontrib><creatorcontrib>Tsygankov, Alexander Y.</creatorcontrib><creatorcontrib>Kunapuli, Satya P.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><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><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dangelmaier, Carol A.</au><au>Patchin, Margaret</au><au>Vajipayajula, Dhruv N.</au><au>Vari, Hymavathi Reddy</au><au>Singh, Pankaj K.</au><au>Wright, Monica N.</au><au>Kostyak, John C.</au><au>Tsygankov, Alexander Y.</au><au>Kunapuli, Satya P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2023-07-01</date><risdate>2023</risdate><volume>299</volume><issue>7</issue><spage>104865</spage><epage>104865</epage><pages>104865-104865</pages><artnum>104865</artnum><issn>0021-9258</issn><eissn>1083-351X</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor–based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>37268160</pmid><doi>10.1016/j.jbc.2023.104865</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0002-2778-2909</orcidid><oa>free_for_read</oa></addata></record> |
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| source | BACON - Elsevier - GLOBAL_SCIENCEDIRECT-OPENACCESS; PubMed Central |
| subjects | Animals Blood Platelets Collection: Signal Transduction hemostasis Mice Phosphorylation platelet Platelet Activation Platelet Aggregation Platelet Membrane Glycoproteins - genetics Platelet Membrane Glycoproteins - metabolism spleen tyrosine kinase Syk Kinase - genetics Syk Kinase - metabolism thrombosis Tyrosine |
| title | Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets |
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