Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at Ser114 contrasts with ERK, which phosphorylated Ser600, and p38, which phosphorylated both sites. Literature reports showed that JNK1/2 activation does not alter eNOS activity and prior work in our lab showed that ERK phosphorylation negativity affects NO output. This thesis presents in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy-Hb) assay. In HMEC-1 cells and in vitro it is observe that phosphorylation at these MAPK sites happens concurrently with activating sites (Ser1177), creating dually phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.
","abstract_html":"<p>Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using <em>in vitro</em> kinase assays and immunoblotting. JNK phosphorylation at Ser<sup>114</sup> contrasts with ERK, which phosphorylated Ser<sup>600</sup>, and p38, which phosphorylated both sites. Literature reports showed that JNK1/2 activation does not alter eNOS activity and prior work in our lab showed that ERK phosphorylation negativity affects NO output. This thesis presents <em>in vitro </em>results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy-Hb) assay. In HMEC-1 cells and <em>in vitro </em>it is observe that phosphorylation at these MAPK sites happens concurrently with activating sites (Ser<sup>1177</sup>), creating dually phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.</p>","abstract_has_math":false,"creators":["Solone, Xzaviar"],"institution":null,"degree_name":"Master of Science in Integrative Biology (MSIB)","degree_level":"Thesis","degree_discipline":"Biology","degree_department":null,"school":null,"contributors":["Dr. Susan Smith","Jonathan McMurry","Dr. Scott Nowak"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-05-23T07:00:00Z","date_published":"2019-05-23T07:00:00Z","updated_at":"2026-07-24T02:43:33Z","subjects":["eNOS","MAP Kinases","nitric oxide synthase","phosphorylation","nitric-oxide","Analytical Chemistry","Biochemistry","Biology","Integrative Biology","Molecular Biology","Structural Biology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.kennesaw.edu/integrbiol_etd/40","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Dr. Susan Smith","Jonathan McMurry","Dr. Scott Nowak"]},{"key":"dc:creator","label":"Author","values":["Solone, Xzaviar"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2021-06-04T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science in Integrative Biology (MSIB)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["eNOS","MAP Kinases","nitric oxide synthase","phosphorylation","nitric-oxide","Analytical Chemistry","Biochemistry","Biology","Integrative Biology","Molecular Biology","Structural Biology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.kennesaw.edu/integrbiol_etd/40"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at Ser114 contrasts with ERK, which phosphorylated Ser600, and p38, which phosphorylated both sites. Literature reports showed that JNK1/2 activation does not alter eNOS activity and prior work in our lab showed that ERK phosphorylation negativity affects NO output. This thesis presents in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy-Hb) assay. In HMEC-1 cells and in vitro it is observe that phosphorylation at these MAPK sites happens concurrently with activating sites (Ser1177), creating dually phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.
"]},{"key":"dc:title","label":"Title","values":["UNDERSTANDING HOW MAP KINASES INFLUENCE ENDOTHELIAL NITRIC-OXIDE SYNTHASE ACTIVITY"]}]}],"canonical_facts":{"dc:contributor":["Dr. Susan Smith","Jonathan McMurry","Dr. Scott Nowak"],"dc:creator":["Solone, Xzaviar"],"dc:date.available":["2021-06-04T07:00:00Z"],"dc:description.abstract":["Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at Ser114 contrasts with ERK, which phosphorylated Ser600, and p38, which phosphorylated both sites. Literature reports showed that JNK1/2 activation does not alter eNOS activity and prior work in our lab showed that ERK phosphorylation negativity affects NO output. This thesis presents in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy-Hb) assay. In HMEC-1 cells and in vitro it is observe that phosphorylation at these MAPK sites happens concurrently with activating sites (Ser1177), creating dually phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.
"],"dc:identifier":["https://digitalcommons.kennesaw.edu/integrbiol_etd/40"],"dc:subject":["eNOS","MAP Kinases","nitric oxide synthase","phosphorylation","nitric-oxide","Analytical Chemistry","Biochemistry","Biology","Integrative Biology","Molecular Biology","Structural Biology"],"dc:title":["UNDERSTANDING HOW MAP KINASES INFLUENCE ENDOTHELIAL NITRIC-OXIDE SYNTHASE ACTIVITY"],"thesis:degree_discipline":["Biology"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science in Integrative Biology (MSIB)"]},"updated_at":"2026-07-24T02:43:33Z"}