Neuronal Nitric-oxide Synthase Is Regulated by the hsp90-based Chaperone System in Vivo

Andrew T Bender,Adam M Silverstein,Damon R Demady,Kimon C Kanelakis,Soichi Noguchi,William B Pratt,Yoichi Osawa

doi:10.1074/jbc.274.3.1472

Andrew T Bender, Adam M Silverstein + Show 5 more

Open Access

https://doi.org/10.1074/jbc.274.3.1472

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Abstract

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR.hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS. hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS. hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.

Highlights

Several transcription factors and protein kinases involved in signal transduction are recovered from cells in association with the ubiquitous heat shock protein hsp901
Neuronal NOS Exists in Native Heterocomplexes with hsp90 —To detect native nNOS1⁄7hsp90 heterocomplexes, cytosol from HEK 293 cells expressing rat neuronal nitric-oxide synthase (nNOS) was immunoadsorbed with nNOS-specific rabbit antiserum, and both the nNOS and the co-immunoadsorbed hsp90 in the washed immune pellet were detected by Western blotting
The native rat nNOS1⁄7human hsp90 heterocomplexes immunoadsorbed from HEK 293 cytosol behaved like steroid receptor1⁄7hsp90 heterocomplexes [1] in that they dissociated when the immune pellets were suspended in buffer and incubated at 25 °C, and this thermal dissociation was inhibited by molybdate

Summary

Introduction

Several transcription factors and protein kinases involved in signal transduction are recovered from cells in association with the ubiquitous heat shock protein hsp901 Complexing of the GR with hsp opens up both thiol moieties [5] and trypsin cleavage sites [6, 7] in the LBD to attack by a thiol-derivatizing agent and the protease These direct data, coupled with recent genetic observations [8], support the notion [9, 10] that the hsp90-based chaperone machinery directs an ATP-dependent partial unfolding of the receptor LBD, making the hydrophobic steroid-binding pocket accessible to steroid. The heme-deficient monomer of NOS can be partially reconstituted in vitro in the presence of heme, tetrahydrobiopterin, and arginine to form the functional homodimer [18, 19]. Garcia-Gardena et al [22] found that hsp was not co-immunoprecipitated with nNOS from lysates of cerebellum, we show here that

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