Breast cancer metastasis suppressor 1 (BRMS1) is stabilized by the Hsp90 chaperone

Abstract

Breast cancer metastasis suppressor 1 (BRMS1) is a member of the mSin3-HDAC transcription co-repressor complex. However, the proteins associated with BRMS1 have not been fully identified. Yeast two-hybrid screen, immuno-affinity chromatography, and co-immunoprecipitation experiments were performed to identify BRMS1 interacting proteins (BIPs). In addition to known core mSin3 transcriptional complex components RBBP1 and mSDS3, BRMS1 interacted with other proteins including three chaperones: DNAJB6 (MRJ), Hsp90, and Hsp70. Hsp90 is a known target of HDAC6 and reversible acetylation is one of the mechanisms that is implicated in regulation of Hsp90 chaperone complex activity. BRMS1 interacted with class II HDACs, HDAC 4, 5, and 6. We further found that BRMS1 is stabilized by Hsp90, and its turnover is proteasome dependent. The stability of BRMS1 protein may be important in maintaining the functional role of BRMS1 in metastasis suppression.

Fig. 1

Identification of chaperone protein interactors. Whole cell lysate from 231-BRMS1 was partially purified with the 3a1.21 mAb that was N-linked to an agarose matrix. Specific fractions of the eluate contained BRMS1 and potential interactors that were combined and electrophoresed on a 12% SDS–PAGE followed by Coomassie staining. Visible bands were excised, trypsin digested, and identified by MALDI-TOF-MS by the UAB MS core facility.

Fig. 2

Validation of BRMS1 interactors. (A) BRMS1 was co-immunoprecipitated with mSDS3, NMI, MRJ, and BAF57 from whole cell lysates (1 mg) of COS7 transient co-transfections. Lane 1, beads with no antibody added, lane 2, whole cell lysate (80 μg), lane 3, immunoprecipitation using an irrelevant antibody (β-actin), lane 4, immunoprecipitation with respective interactor antibody. Western blot (WB) was performed with the ant-901 epitope (BRMS1) antibody. (B) Whole cell lysate (W) from 231-BRMS1 cells were co-immunoprecipitated (IP) with either the BRMS1 3a1.21 mAb or anti-Hsp90 pAb. The blots were probed consecutively with both Abs. S is the supernatant from the IP.

Fig. 3

BRMS1 turnover is proteasome dependent. (A) The half life of BRMS1 is approximately 2 h. The 231-BRMS1 cells were pulsed with media containing [³⁵S]Met for 1 h and collected at selected time points. The lysates were immunoprecipitated with 3a1.21 mAb and the blots were exposed on X-ray film for 14 days. Significant degradation of labeled BRMS1 is noted after 2 h. The blots were re-probed with the 3a1.21 mAb to show no change in total BRMS1. (B) BRMS1 responds to proteasome inhibition. The 231-BRMS1 cells were treated with 20 μM MG-132 (proteasome inhibitor) and compared to the controls (DMSO and untreated). A significant increase in BRMS1 levels is noted in the MG-132 treated cells demonstrating that BRMS1 protein is degraded, probably through proteasomal degradation.

Fig. 4

BRMS1 is stabilized by Hsp90. Inhibition of Hsp90 promotes BRMS1 degradation which is rescued by inhibition of the proteasome. The 231-BRMS1 cells were treated with selected concentrations of the Hsp90 inhibitor geldanamycin (GA) alone for 6 h or in combination with 10 μM MG-132. Significant degradation is noted for BRMS1 in the GA only treated cells. The same blot was probed with antibodies against Hsp90 and β-actin for comparison.

Fig. 5

BRMS1 interacts with class I and class II HDACs. (A) Immunoprecipitation (IP) of BRMS1 from transiently co-transfected COS7 cells and (B) IP of indicated HDAC with anti-FLAG M2 Ab. Class I HDACs, HDAC1 and 2 (represented as H1 and H2) and class II HDACs, HDAC4, 5, and 6 (represented as H4, H5, and H6) were immunoprecipitated by BRMS1 (anti-901 mAb) from whole cell lysates (1 mg) of COS7 cells after transient co-transfections. The top panel in (A) and (B) is whole cell lysate (50 μg) showing levels of various HDACs or BRMS1 after transient transfection. Lane M indicates lane corresponding to the molecular weight marker.