Function and Activation Mechanism of PLEKHG2, A Novel G Beta Gamma-Activated RhoGEF in ...

131 Pages·2016·3.4 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2013
Function and Activation Mechanism of
PLEKHG2, A Novel G Beta Gamma-Activated
RhoGEF in Leukemia Cells
Caitlin M. Runne
University of Iowa
Copyright 2013 Caitlin Marie Runne
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/4907
Recommended Citation
Runne, Caitlin M.. "Function and Activation Mechanism of PLEKHG2, A Novel G Beta Gamma-Activated RhoGEF in Leukemia
Cells." PhD (Doctor of Philosophy) thesis, University of Iowa, 2013.
https://doi.org/10.17077/etd.ab6yvevl
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of thePharmacology Commons
FUNCTION AND ACTIVATION MECHANISM OF PLEKHG2, A NOVEL G -
ACTIVATED RHOGEF IN LEUKEMIA CELLS
by
Caitlin M. Runne
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Pharmacology
in the Graduate College of
The University of Iowa
1
August 2013
Thesis Supervisor: Assistant Professor Songhai Chen
Copyright by
CAITLIN M. RUNNE
2013
All Rights Reserved
2
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Caitlin M. Runne
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Pharmacology at the August 2013 graduation.
Thesis Committee: ___________________________________
Songhai Chen, Thesis Supervisor
___________________________________
Rory A. Fisher
___________________________________
Ernesto J. Fuentes
___________________________________
John G. Koland
___________________________________
Curt D. Sigmund
___________________________________
Stefan Strack
To my family: Mom, Dad, Gram, Aileen, Andrew,
Kieran and John, for your patience, love and support.
2

ii
If you wish to understand what Revolution is, call it Progress; and if you wish to
understand what Progress is, call it Tomorrow.
Victor Hugo
Les Misérables
3

iii
ACKNOWLEDGMENTS
First and foremost, I must extend my deepest gratitude to my mentor, Dr. Songhai
Chen. You have gone above and beyond the call of duty in helping me achieve my goals.
Your time, patience, and effort in helping me develop the skills I will need to pursue my
career, both on and off the bench, are greatly appreciated. I must also thank my thesis
committee: Dr. Rory Fisher, Dr. Ernesto Fuentes, Dr. John Koland, Dr. Curt Sigmund,
and Dr. Stefan Strack. Thank you for your encouragement, your critiques, and your time
over the past four years. Also, thank you to Dr. David Sheff for convincing me to pursue
my PhD instead of my Master’s. It’s a decision I will never regret. Finally, a huge thank
you to the departmental administrative staff: Linda Buckner, Sue Griffith, Lisa Ringen,
Vickie Akers, and Kate Bolton. Thank you for making sure I never missed a deadline and
keeping me on my toes the past four years.
To the members of the Chen lab, both past and present, thank you for friendship
and your mentorship. To Dr. Xiaoyun Tang, Dr. Zhizeng Sun, and Dr. Yuanchao Ye
thank you for your willingness to mentor me, and for all the laughs we’ve shared. To our
undergrads: Kacey Mersch, Caitlin Kilkus, Katelyn Meyer, Jonathan Lochner, and
Jasmin Valentin, thank you for making sure we were always full stocked on tips and
PBS! Getting to meet you all and help you start developing your skills as scientists was
one of my favorite experiences the past four years.
4

To my friends who stood by me along the way, thank you for making sure I was
always found a way to laugh and that I left my house every once in a while. To my East
Coast friends: Heather, Sheena, Laura, Kelly, Akshar, Marissa, Russ, Liz, and everyone
else: thank you for making me feel so loved from afar. I couldn’t have made it through
my first year so far away from home without your constant friendship. To my Iowa
friends: Shippy, Katie, Lauren, Jeanine, Ben, Lightner, Zac, Nick, Justin, Fabian, Simone,
Van, Javier, Edwin, Adele, Beth, Ted, and Audrey: thank you for making sure I
iv
occasionally left the lab, stopped working, and had some fun. I’m so thankful for the
friendships we’ve developed the last four years, and will continue to have in the coming
years.
Most importantly, I have to thank my family. Mom: thank you for not changing
the phone number, despite repeated threats. On a more serious note, I will honestly never
be able to express how grateful I am for everything you’ve done for me the past twenty-
six years. Your constant support, encouragement, and love in every part of my life are the
reasons I have made it as far as I have today. You are the best role model a girl could ask
for. Dad, thank you for your support, love, and for picking me up from the airport at the
strangest hours. To my sister, Aileen, and my brothers, Andrew and Kieran, I am so
proud to be your sister. Aileen, thank you for always being there to talk, no matter how
crazy the hour or how ridiculous the conversation. Andy and Kieran, thank you for
keeping me endlessly entertained and making sure I never take myself too seriously. To
Gram, thank you for always helping me keep a level head. I love you more. Aunt Kathy
and Uncle Brian, thank you for always being one hundred percent behind my decisions
and giving me so much love and support. It means the world.
Finally, to John: thank you for the best year and a half of my life. Who would
have thought the guy who tried to sell me his scooter in Cell Cycle Control class would
be the one? Thank you for being my rock through this entire crazy thesis process, for
5
sitting up every late night while I typed and always making sure I had a fresh cup of
coffee. I couldn’t have done this without you. I promise to be just as supportive when
you write your thesis. Thank you for loving me through it all- I can’t wait to see where
the rest of our lives take us.
v
ABSTRACT
The Rho family of GTPases plays a crucial role in the regulation of diverse
cellular processes, including proliferation and actin cytoskeletal rearrangement to
promote cell migration. However, dysregulation of RhoGTPases has been associated with
disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely
mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or
overexpression contributes to disease pathogenesis. RhoGTPases are activated through
Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have
been identified that activate the 25 RhoGTPases, the pathological role of the majority of
these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated
through tyrosine phosphorylation, a small subset can be activated through heterotrimeric
G proteins, including through G subunits. However, the mechanism by which G
induces RhoGEF activation remains unclear.
PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene
upregulated in a leukemia mouse model, and later shown to be activated by
heterotrimeric G protein subunits. However, its function and activation mechanisms
remain elusive. Here we show that, as compared to primary human T cells, the expression
6

of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by
siRNAs specifically inhibited G -stimulated Rac and Cdc42, but not RhoA activation.
Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion
formation, and leukemia cell migration in response to SDF1 . Additional studies indicate
that G likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This
interaction results in the release of autoinhibition imposed by the C-terminus within a
v i
region encompassing the catalytic DH domain. As a result, overexpressing either the N-
terminus of PLEKHG2 that binds G or the C-terminus that autoinhibits PLEKHG2
blocked G -stimulated Rac and Cdc42 activation and the ability of leukemia cell to
form membrane protrusions and to migrate. Together, our results have demonstrated that
PLEKHG2 functions as a novel G -stimulated RhoGEF that could contribute to
chemokine-induced leukemia cell dissemination and leukemia pathogenesis.
7

vi i