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Understanding How Map Kinases Influence Endothelial Nitric-Oxide Synthase Activity" (2019)

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Understanding How Map Kinases Influence Endothelial Nitric-Oxide Synthase Activity Kennesaw State University DigitalCommons@Kennesaw State University Department of Ecology, Evolution, and Organismal Master of Science in Integrative Biology Theses Biology Fall 5-23-2019 UNDERSTANDING HOW MAP KINASES INFLUENCE ENDOTHELIAL NITRIC- OXIDE SYNTHASE ACTIVITY Xzaviar Solone Follow this and additional works at: https://digitalcommons.kennesaw.edu/integrbiol_etd Part of the Analytical Chemistry Commons, Biochemistry Commons, Integrative Biology Commons, Molecular Biology Commons, and the Structural Biology Commons Recommended Citation Solone, Xzaviar, "UNDERSTANDING HOW MAP KINASES INFLUENCE ENDOTHELIAL NITRIC-OXIDE SYNTHASE ACTIVITY" (2019). Master of Science in Integrative Biology Theses. 40. https://digitalcommons.kennesaw.edu/integrbiol_etd/40 This Thesis is brought to you for free and open access by the Department of Ecology, Evolution, and Organismal Biology at DigitalCommons@Kennesaw State University. It has been accepted for inclusion in Master of Science in Integrative Biology Theses by an authorized administrator of DigitalCommons@Kennesaw State University. For more information, please contact [email protected]. UNDERSTANDING HOW MAP KINASES INFLUENCE ENDOTHELIAL NITRIC-OXIDE SYNTHASE ACTIVITY By Xzaviar Kaymar Victor Solone A Thesis Presented in Partial Fulfillment of Requirements for the Master of Science in Integrative Biology for the Department of Molecular and Cellular Biology Kennesaw State University 1000 Chastain Road Kennesaw, GA 30144 May 2019 _________________________________________________ Dr. Carol Chrestensen, Thesis Chair Professor Department of Chemistry and Biochemistry Kennesaw State University _________________________________________________ Dr. Jonathan McMurry Professor Department of Molecular and Cellular Biology Kennesaw State University _________________________________________________ Dr. Susan Smith Professor Department of Molecular and Cellular Biology Kennesaw State University _________________________________________________ Dr. Scott Nowak Associate Professor Department of Molecular and Cellular Biology Kennesaw State University DEDICATION This thesis is dedicated to my baby nephew Josiah Jacob Solone. He represents the future and has provided me with unconditional motivation to help pave a way for other to pursue STEM- related careers. ii Understanding How Map Kinases Influence Endothelial Nitric-Oxide Synthase Activity 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. Keywords: eNOS, MAP Kinases, nitric oxide synthase, phosphorylation, nitric-oxide iii TABLE OF CONTENTS Dedication ..................................................................................................................ii Abstract ......................................................................................................................iii Table of Contents .......................................................................................................iv List of Figures & Tables ............................................................................................vi Chapter 1: Literature Review ................................................................................1 Significance of Research 1 The Discovery of Nitric-Oxide as a Signaling Molecule 2 Nitric-Oxide Synthases 3 Endothelial Nitric-Oxide Synthase 5 Kinase Regulation of eNOS 6 Hypothesis and Specific Aims ...................................................................................11 Chapter 2: MAP Kinases Recognition Sequences in Human eNOS ...................12 Introduction 12 Experimental Procedures 13 Results 15 Discussion 17 Chapter 3: Understanding how MAP Kinases effect eNOS Activity through Differential Phosphorylation ...............................................................19 Introduction 19 Experimental Procedures 21 Results 25 Discussion 27 Chapter 4: eNOS is Modified in Cells through Multiple Signaling Outputs .....31 Introduction 31 Experimental Procedures 32 Results 34 Discussion 35 Chapter 5: Conclusions, Future Directions, Integrative Statement, and Acknowledgments .................................................................................37 Integrative Statement 38 iv Acknowledgments 39 References ..................................................................................................................40 Supplementary Data ...................................................................................................47 v LIST OF FIGURES AND TABLES LIST OF FIGURES 1. Pathways involved in the synthesis of nitric oxide ..............................................2 2. Schematic overview of NOS structure .................................................................4 3. Sequence alignments of the zinc-binding motif...................................................5 4. Schematic overview of MAPK pathways ............................................................8 5. Graphical Hypothesis ...........................................................................................11 6. NOS multiple sequence alignment.......................................................................14 7. NOS modeling cartoon depicting nitric-oxide synthase ......................................16 8. Measurement of the distances between kinases ...................................................17 9. Schematic representation of hemoglobin species ................................................20 10. Purified eNOS absorbance spectrum from Hi Control ........................................26 11. MAP kinase differently phosphorylates eNOS ....................................................27 12. ERK inhibits eNOS activity while JNK does not ................................................28 13. ERK and P38 do not significantly alter the activity of eNOS .............................28 14. Bovine endothelial nitric-oxide synthase .............................................................30 15. Graphical representation of dually phosphorylated eNOS ..................................31 16. ERK binds differentially phosphorylated eNOS..................................................34 17. eNOS is phosphorylated through multiple signaling outputs ..............................35 LIST OF TABLES 1. eNOS phosphorylation sites and kinases .............................................................7 2. A list of measurements between the kinases docking site ...................................18 3. Buffers used for the purification of eNOS ...........................................................22 4. NOS isolated from diverse species .....................................................................47 vi Chapter 1: Literature Review SIGNIFICANCE OF RESEARCH Cardiovascular Disease According to The Center for Disease Control and Prevention (CDC), approximately 90 million people have known cardiovascular disease. Of these, 800,000 people have heart attacks and over 500,000 people die from heart disease every year (1, 2). One cardiovascular disease is atherosclerosis, the hardening and narrowing of arteries. Usually, atherosclerosis is asymptomatic. However, by the time the disease becomes symptomatic, the arteries have constricted to the point of creating a life-threatening situation. Type 2 Diabetes Mellitus In 2015, the American Diabetes Association (ADA) reported that more than 30 million Americans have been diagnosed with diabetes; this is approximately 9% of the population. The CDC states that Type-2 Diabetes Mellitus (T2DM) accounts for 90 to 95 percent of all diagnosed cases (3). T2DM results from the loss or dampened function of beta-cells to secrete enough insulin to maintain normal blood glucose levels. This results in cellular insulin resistance via hyperinsulinemia (increased insulin secretion due to hyperglycemia; increased blood glucose). People who are insulin resistant undergo phenotypic changes that result in glucose abnormalities such as impaired trafficking of glucose into peripheral tissue (4). Normally, glucose enters the blood-stream via the liver and digestive system; the pancreas secretes insulin which is then recruited to the insulin receptor; promoting glucose
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