Platelet-derived Growth Factor-Mediated Regulation of Slingshot Phosphatase Activity in Vascular Smooth Muscle Cells Open Access

Maheswaranathan, Mithu (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/5138jf27h?locale=en
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Abstract

Abstract
Platelet-derived Growth Factor-Mediated Regulation of Slingshot Phosphatase Activity
in Vascular Smooth Muscle Cells
By Mithu Maheswaranathan

Vascular smooth muscle cell (VSMC) migration contributes to the development of cardiovascular diseases, including atherosclerosis and postangioplasty restenosis. Platelet-derived growth factor (PDGF) mediated VSMC migration requires the NADPH oxidase-1 (Nox1) dependent activation of Slingshot (SSH1L) phosphatase, which dephosphorylates and activates cofilin, a key protein in the regulation of actin dynamics. The overall goal of this project was to investigate the specific mechanism of PDGF-induced SSH1L activation in VSMCs, which is mostly unknown. Because SSH1L has been shown to interact with 14-3-3 regulatory proteins in other cell systems, we hypothesized that 14-3-3 plays a role in SSH1L activation in VSMCs. We demonstrated that the 14-3-3 gamma and beta isoforms form a complex with SSH1L in VSMCs in basal conditions, and that these complexes are disrupted by PDGF treatment, shown by co-immunoprecipitation followed by western blotting. We observed that PDGF induced serine dephosphorylation of SSH1L in a consensus 14-3-3 binding motif in VSMCs derived from wild type, but not Nox1 knockout, mice. We therefore sought to characterize potential phosphorylation sites within the Slingshot protein using site-directed mutagenesis to generate phosphomimetic mutations (serine to aspartic acid) and phosphodeficient mutations (serine to alanine) at position 834. We expressed the SSH1L S834A mutant in VSMC and confirmed that it acts as a constitutively active phosphatase and prevents the ability of PDGF to further regulate the system. Our results demonstrate that the dephosphorylation of SSH1L in a 14-3-3 binding motif site via Nox1-mediated signaling is part of the mechanism of PDGF-induced activation of SSH1L in VSMC, and suggests that disruption of a SSH1L/14-3-3 inhibitory complex is involved. The phosphorylation mutants we generated will be a key tool to further study the specific regulation of SSH1L. These results help provide insight into mechanisms of regulation of VSMC migration, an important event in the development of cardiovascular pathology.

Table of Contents

Table of Contents

Introduction...1-11

Materials and Methods...12-16

a. Cell Culture...12
b. Total Cell Lysate Preparation...12
c. Western Blot...13
d. Immunoprecipitation...13
e. Co-immunoprecipitation...14
f. Site-directed Mutagenesis...14
g. Transfection...14
h. Migration Assay...15
i. Phosphatase Activity Assay...15
j. siRNA Experiments...16

Results...17-34

a. PDGF-induced SSH1L phosphatase activity requires Nox1...17
b. SSH1L acts as a PDGF-sensitive cofilin phosphatase...18
c. PDGF induces SSH1L/14-3-3 complex disruption...20
d. PDGF-induced dephosphorylation of SSH1L is lost in Nox1 KO cells...23
e. 14-3-3 KE mutants may not have displaced endogenous SSH1L...25
f. Generation of SSH1L SA and SD Mutants...28
g. S834A-SSH1L acts as a constitutively active enzyme...31
h. Migration Assay...33

Discussion...35-41

References...42-45

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