Serine phosphorylation differentially affects RhoA binding to effectors: implications to NGF-induced neurite outgrowth.
Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine(188), which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine(188) represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine(188) prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoA(S188A) mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA(14V) does, indicating the requirement of this phosphorylation site. Replacement of serine(188) with the phosphomimetic glutamate residue in RhoA(V14/S188E) selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine(188) may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA.
Nusser
Nóra
N
Department of Physiology, University of Tennessee Health Science Center, Memphis, 894 Union Ave, Memphis, TN 38163, USA.
Gosmanova
Elvira
E
Makarova
Natalia
N
Fujiwara
Yuko
Y
Yang
Linda
L
Guo
Fukun
F
Luo
Yongneng
Y
Zheng
Yi
Y
Tigyi
Gábor
G
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D012694:Serine / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D012694:Serine; D006160:Guanosine Triphosphate; D020932:Nerve Growth Factor; D017868:Cyclic AMP-Dependent Protein Kinases; D020742:rhoA GTP-Binding Protein