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MYO1C Binds to Pip2 with High Affinity Through a Putative Ph Domain

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MYO1C Binds to Pip2 with High Affinity Through a Putative Ph Domain University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Winter 2007 MYO1C Binds to PiP2 With High Affinity Through a Putative Ph Domain David Hokanson University of Pennsylvania Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Medical Biochemistry Commons Recommended Citation Hokanson, David, "MYO1C Binds to PiP2 With High Affinity Through a Putative Ph Domain" (2007). Publicly Accessible Penn Dissertations. 1. https://repository.upenn.edu/edissertations/1 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1 For more information, please contact [email protected]. MYO1C Binds to PiP2 With High Affinity Through a Putative Ph Domain Abstract Myo1c is a member of the myosin superfamily that links the dynamic actin cytoskeleton to the membrane, and plays roles in mechano-signal transduction and membrane trafficking.e W located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. We found that the tail domain binds tightly and specifically ot PIP2 in a non-cooperative manner. It binds with slightly higher affinityo t Ins(1,4,5)P3 as well as other inositol phosphates which may act as inhibitors to membrane binding in the cell. By sequence and secondary structure analysis, we identified this phosphatidylinositol binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for phosphatidylinositol binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro and eradicate correct localization and membrane binding in vivo. The extended sequence of this binding site is conserved within many other myosin-Is across species, suggesting they also contain a putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphatidylinositol specific, but binds anionic phospholipids in a Ca2+ dependent manner; nevertheless, this site is not essential for in vivo membrane binding. As a result, we have determined that myo1c contains two lipid binding sites, a polybasic region that binds to high levels of PS in a Ca2+ dependent manner and a putative PH domain that binds tightly and specifically ot phosphatidylinositols. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor E. Michael Ostap Subject Categories Medical Biochemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1 MYO1C BINDS TO PIP2 WITH HIGH AFFINITY THROUGH A PUTATIVE PH DOMAIN David Hokanson A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2007 _______________________________ Dr. E. Michael Ostap Supervisor of Dissertation _______________________________ Dr. Josh Wand Graduate Group Chairperson ABSTRACT MYO1C BINDS TO PIP2 WITH HIGH AFFINITY THROUGH A PUTATIVE PH DOMAIN David Hokanson Dr. E. Michael Ostap Myo1c is a member of the myosin superfamily that links the dynamic actin cytoskeleton to the membrane, and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. We found that the tail domain binds tightly and specifically to PIP2 in a non-cooperative manner. It binds with slightly higher affinity to Ins(1,4,5)P3 as well as other inositol phosphates which may act as inhibitors to membrane binding in the cell. By sequence and secondary structure analysis, we identified this phosphatidylinositol binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for phosphatidylinositol binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro and eradicate correct localization and membrane binding in vivo. The extended sequence of this binding site is conserved within many other myosin-Is across species, suggesting they also contain a putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphatidylinositol specific, but binds anionic phospholipids in a Ca2+ dependent manner; nevertheless, this site is not essential for in vivo membrane binding. As a result, we have determined that myo1c contains two lipid binding sites, a polybasic region that binds to high levels of PS in a Ca2+ dependent manner and a putative PH domain that binds tightly and specifically to phosphatidylinositols. II Preface When I was looking for a graduate school, while finishing my undergrad degrees at Ohio University, I didn’t know what to expect. I was in complete disbelief after I took my GRE that schools were actually going to pay ME to go to school instead of the other way around. Initially, I selected just two schools to apply to based on the protein folding research that was being done by the faculty, and avoiding any of the pompous Ivy-league schools. I was fortunately unaware at the time that the University of Pennsylvania was, in fact, an Ivy-league school, or I would never have applied. My girlfriend at the time hid this from me, and for that I am grateful. When I discovered the truth a fortnight before my interview, I almost cancelled, but instead I reserved my prejudice and came anyway. I was shocked at how collaborative and non-cutthroat Penn was. It seemed to have all the success and prestige I envisioned an Ivy-league school to have, but was also laid back and friendly. My interview went well and I knew this was where I would end up for the next five to seven years of my life. Mercifully, it has only taken me five years to finish my thesis; although, I have never been in a hurry to leave Philly or Penn. The best decision I could have made was working for Dr. Mike Ostap. I was Mike’s first graduate student, yet it seemed as though he had mentored hundreds before me. I truly believe that much of my happiness here at Penn is due to having a great mentor and lab environment. For anyone that has watched the NBC show, “Scrubs”, Mike is my Dr. Cox. He picks on me constantly and never quite gives me that pat on the back I yearn for, but inspires me to work even harder to get it. To me, Mike is much more than just a brilliant boss who has all the III answers and is rarely wrong, he is a truly inspiring human being. I can’t think of a better man to look up to professionally. There are many people at Penn that I owe some part of my sanity to, including the faculty on my committee and in the PMI, fellow graduate students, and Ruth and Angie in the department offices. After my prelim, I was worried about picking such well-established faculty to serve on my thesis committee for fear that they would make my life hell. However, with each committee meeting I learned that their vast knowledge and expertise in my field were indispensable and made my work more forthright, not more grueling. Also, I’ve made some great friends here and without them, finishing graduate school would have been a much more menacing feat. Thank you fellow BMB students. The Ostap lab is smaller than most labs, and I think that was beneficial to me as a beginning graduate student. I would like to thank current and past members of the lab, in particular, Nanyung, Tianming, Joe, and Jennine. Nanyung was a postdoc when I started in the lab and played a hand in training me to do many of the things I now take for granted (I just wish some of her cloning magic wore off on me). Tianming, what isn’t there to say about Tianming. He is an invaluable member of the lab to me and everyone else. Never complaining, always willing to help, and an absolute master of what he does everyday. Thank you Tianming for constantly keeping the lab stocked with protein and helping in every way you can. Joe has given me the much needed comic relief I’ve needed to get through the frustrating days over the years. You’ve IV always been there to ask the stupid questions I’m too embarrassed to ask Mike. Jennine has been a very welcomed new addition to the lab. I’m thrilled there is someone to take over my project at least in part when I depart. I have enjoyed trying to play the role of a mentor and it is humanitarian of you to always laugh at my jokes. And lastly I would like to thank my family and extended family, my friends. Dad, you always said you would buy me a car if I followed in your footsteps and graduated from med school. Well, I’m graduating from the University of Pennsylvania School of Medicine, it’s a technicality I know, but I’ll take what I can get. Thank you for always believing in me and supporting me through undergrad and the rough times since. Liz, I’m so glad we are still as close as ever. Your support at the beginning and now has always meant so much to me. I am ecstatic that you are ecstatic for me. Oh and I guess thanks for the help with Illustrator too. Bryan, you’ve always been a great friend here at Penn: may we continue to brew great beer and times together for many years. And of course a big thank you to my beautiful girlfriend Sarah (Dorkface). I know I’m probably more difficult than you’ve ever imagined, but I hope it has been worth it for you because it certainly has been for me.
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