Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.
It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Hwei-Ming
HM
Peng
Yoshihiro
Y
Morishima
Gary J
GJ
Jenkins
Anwar Y
AY
Dunbar
Miranda
M
Lau
Cam
C
Patterson
William B
WB
Pratt
Yoichi
Y
Osawa
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D047468:Immunoprecipitation; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010169:Palmitic Acids / Q000378:metabolism; D061988:Proteasome Inhibitors; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D003902:Detergents; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D007074:Immunoglobulin G; D007783:Lactones; D018942:Macrolides; D010169:Palmitic Acids; D061988:Proteasome Inhibitors; D025801:Ubiquitin; C067713:lactacystin; D000255:Adenosine Triphosphate; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; C035359:monorden; D000111:Acetylcysteine