The Role of Nox1 and 14-3-3 in the Regulation of Slingshot Phosphatase in Vascular Smooth Muscle Cells Open Access

Fernandez, Zoila Isabel (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/bz60cx14q?locale=en
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Abstract

Abstract

Nox1 meditated Reactive Oxygen Species Induce Oxidation of 14-3-3 cysteine residues in Vascular Smooth Muscle Cells

By Zoila Isabel Fernadez

Vascular smooth muscle cell (VSMC) migration is important during vascular development and contributes to vascular lesion formation. It plays a role in diseases such as atherosclerosis and postangioplasty restenosis. The mechanism regulating migration of this cell type are therefore of great interest. Recent work in our laboratory has shown that platelet-derived growth factor (PDGF) induces VSMC migration by activating the cofilin phosphatase slingshot (SSH1L) via a reactive oxygen species (ROS) mediated mechanism. The exact mechanism of SSH1L activation, however, is not completely understood. Previous studies have revealed that SSH1L is associated with 14-3-3 proteins in an inhibitory complex that is disrupted serine 834 within 14-3-3 binding motif in SSH1L regulates its binding to 14-3-3 proteins. Interestingly, we describe that the phosphatase responsible for the Serine 834 dephosphorylation and therefore phosphatase activation is SSH1L itself through a mechanism that required Nox1-based NAPDH oxidase. In addition, we established that Nox1-mediated 14-3-3 oxidation of cysteine residues leads to an initial SSH1L/14-3-3 complex disruption that allowed SSH1L autodephosphorylation to occur. Because of the key role of 14-3-3 oxidation, we generated cysteine mutants to identify the redox sensitive sites in 14-3-3 that affect its binding capacity.

Table of Contents

Table of Contents


Introduction ...............................................................................................
................................... 1

Cardiovascular Disease ...................................................................................................................... 1

Vascular Smooth Muscle Cells ............................................................................................................. 2

Migration ........................................................................................................................................ 3

Cofilin ............................................................................................................................................ 3

Slingshot ........................................................................................................................................ 4

PDGF Signaling ................................................................................................................................. 5

Control of VSMC Migration by ROS ....................................................................................................... 6

PDGF induces SSH1L activation via a Nox1-dependent pathway ................................................................ 8

14-3-3 Protein and 14-3-3/SSH1L Complex ........................................................................................... 9

Hypothesis .................................................................................................................................. 10

Materials and Methods .................................................................................................................. 11

Cell Culture .................................................................................................................................... 11

Plasmids ........................................................................................................................................ 11

Total Cell Lysate Preparation.............................................................................................................. 11

Transfection ................................................................................................................................... 12

Western Blot .................................................................................................................................. 12

Co-Immunoprecipitation (IP) .............................................................................................................. 13

Phosphatase Activity Assay .............................................................................................................. 14

Site-Directed Mutagenesis ................................................................................................................ 14

Labeling with 5-Iodoacetamido Fluorescein (5-IAF) ................................................................................ 15

Statistical Analysis .......................................................................................................................... 15

Results ......................................................................................................................................... 16

Phosphorylation of Serine 834 site controls SSH1L binding to 14-3-3 ......................................................... 16
SSH1L is autodephosphorylated .......................................................................................................... 18
14-3-3 cysteine residues are oxidized after PDGF treatment which reduces 14-3-3 binding to SSH1L ............... 20
14-3-3 Structure and Generation of 97/98 and 192 Mutants ..................................................................... 24
14-3-3 mutant binding capacity to SSH1L in basal and H2O2-treated conditions (Preliminary).......................... 27
Discussion ..................................................................................................................................... 29
Limitations and Future Directions.......................................................................................................... 32
References..................................................................................................................................... 34

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