Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

Jacob R Manjarrez,Robert L Matts,Thomas Prince,Liang Sun,Didier Picard

doi:10.1371/journal.pone.0090054

Abstract

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes.

Highlights

Excluding skin cancer, breast cancer is the most common cancer, and the second leading cause of cancer deaths among women, with approximately a quarter of a million new cases of breast cancer being diagnosed annually [1]
To verify that DBC2 was a protein that interacts with the Hsp90 chaperone machine, a cDNA I.M.A.G.E clone encoding full length human DBC2 was obtained from the ATCC and cloned into the pcDNA3.1 vector with a FLAG-tag inserted at the N-terminus of the DBC2 coding sequence
Western blot analysis indicated that Hsp90 and Cdc37 co-adsorbed with DBC2, verifying the protein as an interacting partner of the Hsp90/Cdc37 chaperone machine (Fig. 1A)

Summary

Introduction

Breast cancer is the most common cancer, and the second leading cause of cancer deaths among women, with approximately a quarter of a million new cases of breast cancer being diagnosed annually [1]. An additional contributing gene Deleted-in-Breast Cancer 2 (DBC2, a.k.a RhoBTB2) was identified in a region of human chromosome 8p21 that is homologously deleted in 3.5% of breast tumors [4] Loss of this region of chromosome 8 is among the most frequent genetic defects found in prostate cancer [5,6], and has been implicated in other common forms of cancer: ovarian [7], lung [8,9], colorectal [9,10,11], liver [9], bladder [12], and kidney cancers [13]. Leading further support to its role as a tumor suppressor, ectopic expression of wild-type DBC2, but not its mutants, in T-47D breast cancer cells that lack DBC2 expression caused growth inhibition [4]

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Results

Conclusion