Hsp90 phosphorylation, Wee1 and the cell cycle

Abstract

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1∆ yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.

Figure 1.

Model depicting the Hsp90 chaperone cycle. ATP binding to the N-terminal domains of Hsp90 (open) promotes repositioning of a “lid” segment followed by transient dimerization of the N-domains. Subsequent structural rearrangements result in the (closed and twisted) conformation of Hsp90 that is competent for ATP hydrolysis. Binding of the co-chaperone Aha1 enhances Hsp90 ATPase activity. The co-chaperones Sti1/HOP and Cdc37/p50, or pharmacologic inhibitors such as geldanamycin or radicicol, exert an opposite effect by blocking the initial structural changes necessary for N-domain dimerization. Sba1/p23 strengthens the late Hsp90 conformation and inhibits ATP hydrolysis. Domain labeling is as follows: N, N-domain (blue); CL, charged linker (red); M, M-domain (yellow); C, C-domain (green); ATP lid, (purple).

Figure 2.

Yeast Hsp90 phosphorylation on serine (phos-Ser) and threonine (phos-Thr) residues. yHsp90-His₆ was purified from yeast cells that were heat shocked at 39°C for 40 min or treated with 100 μM geldanamycin (GA) for 60 min. wild-type cells containing empty plasmid were used as negative control.

Figure 3.

wee1, an Hsp90 client protein, phosphorylates a conserved tyrosine residue (Y38) in the N-domain of a subpopulation of nuclear-localized yHsp90. Phosphorylation also leads to ubiquitination and degradation of Hsp90 by cytoplasmic proteasomes. Pharmacologic inhibition/molecular silencing of wee1 inhibits Hsp90 chaperoning of distinct clients and sensitizes cells to Hsp90 inhibitor-induced apoptosis. Domain labeling is as follows: N, N-domain (blue); CL, charged linker (yellow); M, M-domain (red); C, C-domain (green); ATP lid, (purple).

Figure 4.

Yeast cells expressing yHsp90-Y24F and swe1Δ cells are hypersensitive to GA. Yeast cells were grown to mid-log and then a 1:10 dilution series were spotted on YPD agar containing 100 μM GA. Plates were incubated at 25°C for 4 days.

Figure 5.

Effect of Hsp90 inhibitors GA or radicicol (RD) on the stability of Swe1 tyrosine kinase. Yeast cells were grown to mid-log and then treated with 50 μM GA or RD. Swe1-HA in yeast lysate was detected by western blot using an anti-HA-monoclonal antibody. yHsp90-His₆ was used as loading control.

Figure 6.

Swe1 destabilization in yHsp90-Y24F-expressing yeast. (A) western blotting was used to detect Swe1-HA in yeast cell lysate expressing either wild-type yHsp90 or yHsp90-Y24F. yHsp90-His₆ was used as loading control. (B) Association of GST-tagged Swe1 with wild-type yHsp90 and yHsp90-Y24F. GST-tagged Swe1 under galactose (gal) inducible promoter (GAL1) was expressed in yeast cells containing either wild-type yHsp90 or yHsp90-Y24F. Swe1-GST co-precipitating with yHsp90-His₆ was detected by western blotting.

Figure 7.

Lack of G₂/M checkpoint-induced delay of nuclear division in yHsp90-Y24F and swe1Δ cells. (A) Flow cytometric analysis of the DNA content of asynchronously growing wild-type, swe1Δ, and yHsp90-Y24F yeast cells. Occupancy of G₂ is unaltered in the two mutants when compared to wild-type cells (wild-type, 48.7%; swe1Δ, 49.0%; yHsp90-Y24F, 51.8%). (B) Cells were released from α-factor-induced cell cycle arrest into fresh medium containing 50 μM Lat-A. inclusion of Lat-A causes arrest at the G₂/M checkpoint. At the indicated times, cell aliquots were removed, fixed and stained with DAPi to visualize DNA, and 100 cells were scored. Premature nuclear division is apparent in both yHsp90-Y24F mutant and swe1Δ cells.