Chaperone-dependent Regulation of Endothelial Nitric-oxide Synthase Intracellular Trafficking by the Co-chaperone/Ubiquitin Ligase CHIP

Abstract

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS⅐Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.The nitric-oxide synthases (NOSs) 1 are a family of mammalian enzymes that catalyze the oxidation of L-arginine to produce NO and L-citrulline. Three NOS isoforms exist in mammalian cells, neuronal (nNOS; NOS1), inducible (iNOS; NOS2), and endothelial (eNOS; NOS3), named after the cell types in which they were originally discovered. All NOS isoforms have similar primary structures, including an oxygenase domain at the N terminus, a reductase domain at the C terminus, and a hinge calmodulin domain in between. eNOS is unique among the NOS isoforms in that it is dually acylated by myristate and palmitate. Cysteine palmitoylation is necessary for targeting of eNOS to the specific plasmalemmal microdomain, caveolae (1), and both fatty acylations are required for specific targeting of eNOS to the Golgi (2). Correct subcellular trafficking and localization to the plasmalemma is necessary for eNOS function. eNOS produces NO (and/or other reactive nitrogen species) in vascular endothelial cells and cardiomyocytes in response to a variety of agonists and mechanical stimuli (i.e. shear) (3, 4). Mislocalization of the enzyme to either domain impairs agonist-stimulated eNOS activation and optimal NO release from cells, implying that the proper subcellular localization of eNOS is critical for optima...

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

“…vascular endothelial growth factor, insulin, and estrogen, would preferentially active Golgi eNOS, and conversely, calcium-dependent agonists such as thapsigargin and bradykinin would preferentially activate plasma membrane eNOS. This concept is strengthened by the recent discoveries of two novel eNOS-binding partners, NOSIP and NOSTRIN (47,48), and an indirect regulator, CHIP (57). Overexpression of both NOSIP and NOSTRIN influences eNOS activity only in vivo by redistributing eNOS from the plasma membrane to intracellular sites.…”

An Androgen Receptor NH2-terminal Conserved Motif Interacts with the COOH Terminus of the Hsp70-interacting Protein (CHIP)

“…CHIP may act as a molecular link between the protein folding chaperone functions and proteasome-mediated protein degradation processes of the Hsp70⅐Hsp90 heterocomplex (52). CHIP also directly modulates the stress response by activating heat shock factor-1 (53) and influences protein trafficking (54).…”

Ubiquitination-mediated protein degradation and modification: an emerging theme in plant-microbe interactions

“…In mammals, the CHIP protein interacts with molecular chaperones such as HSP70 and HSP90 family members and removes substrate proteins of chaperones from the folding pathway to the ubiquitin-proteasome pathway for degradation [68]. CHIP was shown to possess an E3 ubiquitin ligase activity [69,70] and has been implicated in ubiquitination and proteasomal targeting of a variety of proteins, such as transcription factors SMAD and E2A [71,72] and nitric oxide synthase [73,74]. CHIP was also found to be able to promote formation of Lys63-linked polyubiquitin chains in collaboration with Uev1a-Ubc13 in vitro [75].…”