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KINASE ACTIVITY OF THE PSEUDOHYPOALDOSTERONISM TYPE II
GENE PRODUCT, WNK4
by
Erik Robert Ahlstrom
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHYSIOLOGY AND BIOPHYSICS)
May 2008
Copyright 2007 Erik Robert Ahlstrom
Object Description
| Title | Kinase activity of the pseudohypoaldosteronism type II gene product, WNK4 |
| Author | Ahlstrom, Erik Robert |
| Author email | ahlstrom@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Physiology & Biophysics |
| School | Keck School of Medicine |
| Date defended/completed | 2007-11-21 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-01-25 |
| Advisor (committee chair) | Yu, Alan S.L. |
| Advisor (committee member) |
Shen, Wei-Chiang McDonough, Alicia A. Farley, Robert A. Peti-Peterdi, Janos |
| Abstract | Mutations in WNK1 (with no K [lysine] 1) and WNK4 protein kinases cause pseudohypoaldosteronism type II (PHAII), a rare genetic disorder that features high blood pressure and elevated serum potassium levels. Potential targets of WNK4 regulation have been identified by various approaches, but the mechanism by which it influences its targets is still poorly understood. In an effort to characterize its kinase activity, we expressed and purified full-length and truncated forms of WNK4 from HEK293 cells. Due to endogenous kinases binding non-specifically to the protein G resin when immunoprecipitating WNK4 from HEK293 cells, we decided to recover the protein by the tandem affinity purification (TAP) method. Our phosphorylation experiments identified a 40 kD kinase that associates specifically to the C-terminal half of WNK4 and is able to phosphorylate WNK4 and the WNK4 substrate, oxidative stress response kinase 1 (OSR1). This kinase copurifies with WNK4 in the mammalian cell lines HEK293, COS-7 and CHO, but could not be identified by peptide mass fingerprinting. By modifying the TAP protocol to include high salt and detergent washes, we were able to dissociate a large fraction of the 40 kD kinase and measure specific in vitro phosphorylation of OSR1 by full-length and delta593 truncated WNK4. The PHAII associated mutations E559K, D561A and Q562E had no significant effect on this phosphorylation. This study contributes important information on the mechanism by which WNK4 may influence its targets by showing that full-length WNK4 has kinase activity and also what effects the PHAII mutations have on this activity. |
| Keyword | protein kinase; hypertension; pseudohypoaldosteronism type 2 |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m991 |
| Contributing entity | University of Southern California |
| Rights | Ahlstrom, Erik Robert |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@dots.usc.edu |
| Filename | etd-Ahlstrom-20080125 |
| Archival file | uscthesesreloadpub_Volume51/etd-Ahlstrom-20080125.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@dots.usc.edu |
| Full text | KINASE ACTIVITY OF THE PSEUDOHYPOALDOSTERONISM TYPE II GENE PRODUCT, WNK4 by Erik Robert Ahlstrom A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHYSIOLOGY AND BIOPHYSICS) May 2008 Copyright 2007 Erik Robert Ahlstrom |
