HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone and ansamycin class of antibiotic, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, including the kinasesz src, erbB2 and c-raf-1, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. With respect to c-raf-1, no other members of the MAP kinase signalling pathway appear to be affected by GA. With respect to erbB2, it appears to be the most sensitive member of the EGF receptor family of tyrosine kinases to this drug. In the case of mutated p53, attainment of the mutated (but not wild type) conformation of the protein requires transient interaction with HSP90 and an accessory protein termed p23. GA treatment destroys the conformation and the function of mutated p53, providing evidence that pharmacologic manipulation of protein conformation is feasible. In collaboration with Pfizer Central Research, we have tested more than 35 benzoquinone ansamycin derivatives for their ability to bind HSP90/GRP94 and to affect the stability and function of mutant p53, c-raf-l and erbB2. Benzoquinone ansamycins are currently the only agents capable of specifically interfering in HSP90/GRP94 function and these drugs will be useful agents in further studying the role of these chaperones proteins in regulating the stability and function of oncogenes and other signalling proteins.
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