Article thumbnail

Investigating the regulation of protein phosphatase 5 by the monomeric GTPase Rac1

By Anindya Chatterjee

Abstract

Physiological studies performed in rat pituitary cells implicated Protein phosphatase 5 (PP5) as an effector of Rac1 GTPase in the regulation of Kv11.1 ion channel, but direct biochemical evidence for PP5 regulation by Rac1 is lacking. In this study I used immunoprecipitation, in vitro binding, cellular fractionation, and immunofluorescence techniques to show that the tetratricopeptide repeat (TPR) domain of PP5 interacts specifically and directly with active Rac1. Consequently, activation of Rac1 promoted PP5 translocation to the plasma membrane in intact cells and stimulated PP5 phosphatase activity in vitro. In contrast, neither constitutively active RhoA-V14 nor dominant negative Rac1N17, which preferentially binds GDP and retains an inactive conformation, bound PP5 or stimulated its activity. In addition, dominant negative Rac1N17 and Rac1(PBRM), a mutant lacking the C-terminal polybasic region required for Rac1 association with the membrane, both failed to cause membrane translocation of PP5. Mutation of predicted contact residues in the PP5 TPR domain (K93) or within Rac1 (G30) also disrupted co-immunoprecipitation of Rac1:PP5 complexes and membrane translocation of PP5. Specific binding of PP5 to activated Rac1 provides a direct mechanism by which PP5 can be stimulated and recruited to participate in Rac1-mediated signaling pathways. Kv11.1 potassium ion channels are responsible for the rhythmic contraction of cardiac myocytes that depends on its regulation via phosphorylation/dephosphorylation events. Improper regulation of this ion channel results in cardiac arrhythmias and sudden death syndrome. My findings may provide clues for enhancing the understanding of the role of PP5 in this regulation of Kv11.1 channel protein and other disease conditions

Topics: Cellular biology|Biochemistry
Publisher: 'Purdue University (bepress)'
Year: 2010
OAI identifier: oai:docs.lib.purdue.edu:dissertations-9821
Provided by: Purdue E-Pubs
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • https://docs.lib.purdue.edu/di... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.