UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

Abstract

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.

Figure-1. Ubr1−/− cells are less sensitive to treatment with geldanamycin

A. WT and Ubr1^−/− MEF cells were treated with 1µM of GA for different times. 20 µg of total protein from each cell line was fractionated by SDS-PAGE and probed with antisera for total Akt (t-Akt), Cdk4 and Actin. B and C. Quantification of t-Akt (B) and Cdk4(C) levels in extracts from cells treated with GA. n = 5 +/− standard error. Statistical analysis of the difference between WT and UBR1^−/− at a given concentration was calculated by paired t test (* p< .05 and ** p<.005) D. Ubr2^−/− and Ubr1^−/− Ubr2^−/− MEF cells were treated with 1µM of GA for the indicated time points. Extracts were probed with antisera to Akt (t-Akt), Cdk4 and Actin. E. All four MEF cell lines were treated with different concentrations of GA for 24 hours. 20 µg of total protein from each cell line was fractionated by SDS-PAGE and probed for t-Akt, phospho-Akt (pAkt; S473) and Actin.

Figure-1. Ubr1−/− cells are less sensitive to treatment with geldanamycin

A. WT and Ubr1^−/− MEF cells were treated with 1µM of GA for different times. 20 µg of total protein from each cell line was fractionated by SDS-PAGE and probed with antisera for total Akt (t-Akt), Cdk4 and Actin. B and C. Quantification of t-Akt (B) and Cdk4(C) levels in extracts from cells treated with GA. n = 5 +/− standard error. Statistical analysis of the difference between WT and UBR1^−/− at a given concentration was calculated by paired t test (* p< .05 and ** p<.005) D. Ubr2^−/− and Ubr1^−/− Ubr2^−/− MEF cells were treated with 1µM of GA for the indicated time points. Extracts were probed with antisera to Akt (t-Akt), Cdk4 and Actin. E. All four MEF cell lines were treated with different concentrations of GA for 24 hours. 20 µg of total protein from each cell line was fractionated by SDS-PAGE and probed for t-Akt, phospho-Akt (pAkt; S473) and Actin.

Figure-2. Induction of Hsp70 and Hsp90 in Ubr1−/−, Ubr2−/− and double knockout Cells

Cells as indicated were treated with 1µM GA for the times indicated and analyzed by Western blot for the levels of Hsp70 and Hsp90. Phosphatidyl inositol 3 kinase (PI3K) was used as a loading control

Figure-3. Effect of Cycloheximide on Protein Kinase levels in Ubr1−/− Cells

A. Schematic of experimental approach. Cells were treated with GA for 12 hours before subsequent treatment with or without cycloheximide. Aliquots of cells were taken at 15, 18 and 20 hours after initial geldanamycin treatment for Western blot analysis. B. Western blot analysis of total Akt (t-Akt), Cdk4 and actin (loading control).

Figure-4. Effects of the Hsp90 inhibitor PU-H71

A.WT and Ubr1^−/− cells were treated for 24 hours with indicated concentrations of PU-H71 and cell extracts from each cell line analyzed by western blot for t-Akt, p-Akt (S473), Cdk4 and Actin. B. Time course analysis of WT and UBR1^−/− MEF cells after treatment with 500 nM of PU-H71. Western blot for t-Akt, p-Akt (S473), Cdk4 and Raf-1. Actin was used as a loading control.

Figure-5. UBR1 and UBR2 promote sensitivity to Hsp90 inhibitors

A. WT, Ubr1^−/−, Ubr2^−/− and Ubr1^−/− Ubr2^−/− cells were treated with indicated concentrations of GA for 24 hours and cell viability was measured as described in materials and methods. B. Same as A except the Hsp90 inhibitor used was PU-H71.C. Ubr1^−/− cells were mock transfected (grey bars) and tranfected with a plasmid encoding rat Ubr1 (rUbr1; black bars). After 22 hours of transfection, cells were treated with different concentrations of GA for 24 hours and cell viability was analyzed as in A. Experiments were performed in quadruplicate and the bars represent the mean from three independent experiments. Error bars indicate standard error. Statistical analysis of the difference between WT and UBR1^−/−, UBR2^−/−, Ubr1^−/− Ubr2^−/− (A and B), mock and rUbr1 transfected cells (C) at a given concentration was calculated by paired t test (* p<.05 and ** p<.005).

Figure-6. Effects of UBR1 knockdown in the breast cancer cell line BT474 and wild type MEF cells upon Hsp90 inhibition

A. BT474 cells were stably transfected with control and UBR1 shRNA. Transfected cells were treated with 250 nM GA for 0, 6, 12, 18, 24, and 36 hours. Cell extracts were probed with antisera to p-Akt (Ser 473), Akt, Cdk4, ErbB2, UBR1 and Actin. B. Effect of siRNA knockdown of Ubr1 in MEF cells. Panels show Western blot analysis of total Akt (t-Akt), Ubr1 and actin as a loading control after 24 hours of GA treatment at the concentrations indicated. C. Quantification of the levels of total Akt in control and Ubr1 siRNA treated cells after geldanamycin treatment. N =3 +/− SE; *p <0.05.

Figure-7. Functional relationship between CHIP and UBR1

A. WT and Ubr1^−/− cells were transfected with control and CHIP siRNA. After 22 hours of transfection, cells were treated with different concentrations of GA and DMSO for 24 hours. 20 µg of total proteins was fractionated by SDS-PAGE and probed with CHIP, Akt, pAkt, Cdk4, and Actin. B. Quantification of t-Akt normalized against Actin. The bars show the remaining amounts of t-Akt after GA treatment in 3 independent experiments. Error bars indicate the standard error (SE). C. Quantification of Cdk4 level. Details as in B. Statistical analysis of the difference between Ubr1^−/− plus Control siRNA and Ubr1^−/− +CHIP siRNA at a given concentration was calculated by unpaired t test (* p< .05, ** p<.005 and *** p<.0005).