Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress.
The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.
Khanday
Firdous A
FA
Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA.
Yamamori
Tohru
T
Mattagajasingh
Ilwola
I
Zhang
Zhe
Z
Bugayenko
Artem
A
Naqvi
Asma
A
Santhanam
Lakshmi
L
Nabi
Nusrat
N
Kasuno
Kenji
K
Day
Billy W
BW
Irani
Kaikobad
K
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D051379:Mice; D018384:Oxidative Stress; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013912:Threonine; D012694:Serine; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein