Dynamic cycling with Hsp90 stabilizes neuronal nitric oxide synthase through calmodulin-dependent inhibition of ubiquitination.
NO production by neuronal nitric oxide synthase (nNOS) requires calmodulin and is enhanced by the chaperone Hsp90, which cycles dynamically with the enzyme. The proteasomal degradation of nNOS is enhanced by suicide inactivation and by treatment with Hsp90 inhibitors, the latter suggesting that dynamic cycling with Hsp90 stabilizes nNOS. Here, we use a purified ubiquitinating system containing CHIP (carboxyl terminus of Hsp70-interacting protein) as the E3 ligase to show that Hsp90 inhibits CHIP-dependent nNOS ubiquitination. Like the established Hsp90 enhancement of NO synthesis, Hsp90 inhibition of nNOS ubiquitination is Ca2+/calmodulin-dependent, suggesting that the same interaction of Hsp90 with the enzyme is responsible for both enhancement of nNOS activity and inhibition of ubiquitination. It is established that CHIP binds to Hsp90 as well as to Hsp70, but we show here the two chaperones have opposing actions on nNOS ubiquitination, with Hsp70 stimulating and Hsp90 inhibiting. We have used two mechanism-based inactivators, guanabenz and NG-amino-L-arginine, to alter the heme/substrate binding cleft and promote nNOS ubiquitination that can be inhibited by Hsp90. We envision that, as nNOS undergoes toxic damage, the heme/substrate binding cleft opens exposing hydrophobic residues as the initial step in unfolding. As long as Hsp90 can form even transient complexes with the opening cleft, ubiquitination by Hsp70-dependent ubiquitin E3 ligases, like CHIP, is inhibited. When unfolding of the cleft progresses to a state that cannot cycle with Hsp90, Hsp70-dependent ubiquitination is unopposed. In this way, the Hsp70/Hsp90 machinery makes the quality control decision for stabilization versus degradation of nNOS.
Peng
Hwei-Ming
HM
Department of Pharmacology, the University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
Morishima
Yoshihiro
Y
Clapp
Kelly M
KM
Lau
Miranda
M
Pratt
William B
WB
Osawa
Yoichi
Y
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D003500:Cyclization; D019281:Dimerization; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects*
D002147:Calmodulin; D018841:HSP90 Heat-Shock Proteins; D025801:Ubiquitin; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I