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A Study of the Protein Phosphatase 1 (PP1) Regulatory Subunit, TIMAP, in Human Glomerular Endothelial Cells Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Obeidat, Marya Mr
Supervisor and department
Ballermann, Barbara (Medicine)
Examining committee member and department
Goping, Ing Swie (Biochemistry)
Muruve, Daniel (Medicine, University of Calgary)
Brindley, David (Biochemistry)
Holmes, Charles (Biochemistry)
Eitzen, Gary (Cell Biology)
Department of Medicine
Experimental Medicine
Date accepted
Graduation date
Doctor of Philosophy
Degree level
TIMAP is a prenylated endothelial-predominant regulatory subunit for the serine/threonine (Ser,S/Thr,T) protein phosphatase 1 catalytic subunit (PP1c). TIMAP was first discovered in glomerular endothelial cells. It is expressed in the developing blood vessels of kidney, and its transcription in cultured glomerular endothelial cells is downregulated by the transforming growth factor beta 1 (TGFβ1). In turn, TGFβ1 is a critical regulator of endothelial cell development and differentiation. According to its amino acid sequence, TIMAP belongs to the family of myosin phosphatase regulatory subunits (MYPT), which regulate myosin II dynamics required for various cellular processes including cell proliferation, adhesion, migration and endothelial blood barrier function. In this thesis, we explored the functional role of TIMAP in the regulation in glomerular endothelial cells. We demonstrated for the first time that TIMAP protein depletion attenuates glomerular endothelial cell proliferation, survival and sprouting angiogenesis. We found that TIMAP protein expression is required to maintain the activating phosphorylation of the Ser/Thr kinase Akt, a major positive regulator of cell survival, proliferation, and angiogenesis. Also, we discovered that TIMAP protein depletion reduces the inhibitory Ser 370 phosphorylation of the phospholipid phosphatase and tumor suppressor PTEN, a negative regulator of Akt activity, cell survival, proliferation and angiogenesis. Since TIMAP belongs to the family of myosin phosphatase targeting subunits, we also explored whether it regulates phosphorylation of the myosin II regulatory light chain (MLC2) in vitro and in vivo. We show that the TIMAP/PP1c holoenzyme can effectively dephosphorylate pMLC2 in vitro, but that this function is not observed in living cells, given that TIMAP overexpression strongly augmented MLC2 phosphorylation instead. The TIMAP-induced MLC2 phosphorylation in the glomerular endothelial cells was indirect, and required the TIMAP-PP1c association. The TIMAP-mediated MLC2 hyperphosphorylation was due to a markedly reduced rate of MLC2 dephosphorylation. Since MYPT1 is the predominant MLC2 phosphatase in endothelial cells, we explored whether TIMAP/PP1c inhibits the MYPT1/PP1c phosphatase. TIMAP did not suppress MYPT1 or PP1c expression, it did not bind MYPT1 directly, nor did it alter RhoA- or CP17-dependent MYPT1 regulation. The mechanism whereby TIMAP overexpression reduces MLC2 dephosphorylation therefore remains obscure. Finally, we observed that MLC2 and pMLC2 can bind TIMAP directly, and that this interaction blocks the activating kinase-mediated phosphorylation of MLC2 in vitro. We conclude that TIMAP/PP1c can regulate MLC2 phosphorylation in glomerular endothelial cells, but that this effect is not due to direct myosin phosphatase activity in the living cell.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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