The function and regulation of vinculin in cell-cell adhesions

185 Pages·2016·6.66 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
2011
The function and regulation of vinculin in cell-cell
adhesions
Xiao Peng
University of Iowa
Copyright 2011 Xiao Peng
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/2605
Recommended Citation
Peng, Xiao. "The function and regulation of vinculin in cell-cell adhesions." PhD (Doctor of Philosophy) thesis, University of Iowa,
2011.
https://doi.org/10.17077/etd.1zegjhiv.
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theBiochemistry Commons
THE FUNCTION AND REGULATION OF VINCULIN IN CELL-CELL ADHESIONS
by
Xiao Peng
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Biochemistry
in the Graduate College of
The University of Iowa
May 2011
Thesis Supervisor: Assistant Professor Kris A. DeMali
1

ABSTRACT
Adherens junctions are essential for embryogenesis and tissue homeostasis. The
major transmembrane adhesion receptors in adherens junctions are the cadherins, which
mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is
regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one
cytoplasmic component of the cadherin adhesion complex, but unlike other junction
components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cell-
matrix adhesions has commanded the most attention, while little is known about its role
in cell-cell adhesions.
To define the role of vinculin in adherens junctions, I established a short hairpin
RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites
of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology
was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell
surface. I investigated the mechanism for this effect and found that vinculin must bind to
β-catenin to regulate E-cadherin surface expression.
Having established a role for vinculin in cell-cell adhesions, the critical question
became how vinculin recruitment to and activation at cell-cell junctions are regulated. I
found that α-catenin triggers activating vinculin conformational changes. Unlike all of
the known vinculin activators in cell-matrix adhesions, α-catenin binds and activates
vinculin independently of an A50I substitution. Thus, adherens junction activators and
cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using
mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment
to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822.
Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly
of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially
recruited to adherens junctions and cell-matrix adhesions and describes the first known
2

role for vinculin at cell-cell adhesions.

Abstract Approved: _________________________________________
Thesis Supervisor
_________________________________________
Title and Department
_________________________________________
Date
THE FUNCTION AND REGULATION OF VINCULIN IN CELL-CELL ADHESIONS
by
Xiao Peng
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Biochemistry
in the Graduate College of
The University of Iowa
May 2011
Thesis Supervisor: Assistant Professor Kris A. DeMali
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
Xiao Peng
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Biochemistry at the May 2011 graduation.
Thesis Committee: ___________________________________
Lori Wallrath, Thesis Chair
___________________________________
Jim Lin
___________________________________
David Price
___________________________________
Peter Rubenstein
___________________________________
Daniel Weeks
To my parents
ii
ACKNOWLEDGMENTS
I would like to thank my parents for all of their support. I could not have
accomplished this without them. Special thanks to my thesis advisor Dr. Kris DeMali for
her instruction, encouragement and patience throughout these years. Her understanding
and support guided me through the ups and downs of my graduate career. To all current
and past members of the DeMali lab, Elke, Jess, Jen, Xiaowen, Chaoqun, Laura, Liz,
Andrew, Amy, Cort, Ashley, and Melissa: thank you all so much. I have formed special
and wonderful friendships with all of you. I would also like to thank the graduate
students and postdocs in the Biochemistry Department, especially Kuo-Kuang, Tyson and
Xu for your help and friendship. Last and not the least, I would like to thank my thesis
committee members for your invaluable suggestions and discussions.
iii
ABSTRACT
Adherens junctions are essential for embryogenesis and tissue homeostasis. The
major transmembrane adhesion receptors in adherens junctions are the cadherins, which
mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is
regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one
cytoplasmic component of the cadherin adhesion complex, but unlike other junction
components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cell-
matrix adhesions has commanded the most attention, while little is known about its role
in cell-cell adhesions.
To define the role of vinculin in adherens junctions, I established a short hairpin
RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites
of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology
was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell
surface. I investigated the mechanism for this effect and found that vinculin must bind to
β-catenin to regulate E-cadherin surface expression.
Having established a role for vinculin in cell-cell adhesions, the critical question
became how vinculin recruitment to and activation at cell-cell junctions are regulated. I
found that α-catenin triggers activating vinculin conformational changes. Unlike all of
the known vinculin activators in cell-matrix adhesions, α-catenin binds and activates
vinculin independently of an A50I substitution. Thus, adherens junction activators and
cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using
mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment
to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822.
Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly
of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially
recruited to adherens junctions and cell-matrix adhesions and describes the first known
iv
role for vinculin at cell-cell adhesions.
v