via association with the F box containing ubiquitin ligases. We have ruled out the possibility that the observed down regulation of Wee1 was caused by mitosis induced by 17AAG. First, checkpoint competent parental HCT116 cells treated SRT1720 sequentially with SN 38 followed by 17AAG remained arrested in G2 without mitotic entry, and yet Wee1 expression declined markedly in these cells . Second, in checkpoint defective HCT116 p53 null cells, the loss of Wee1 and Chk1 upon sequential treatment with SN 38 and 17AAG preceded the activation of the promitotic cyclin B1 associated kinase by 6 h. Together, these results strongly suggest that the depletion of Wee1 after 17AAG treatment is a direct effect of Hsp90 inhibition rather than a consequence of mitotic entry induced by the drug.
Kaempferol In addition, we examined the level of Myt1, another key regulatory kinase of G2 M transition, after sequential treatment with SN 38 and 17AAG in HCT116 p53 null cells. In contrast to Chk1, the protein level of Myt1 was not appreciably affected by SN 38 and 17AAG. At a late time point, there was an upward motility shift of Myt1, consistent with the mitotic form of this protein reported in the literature and, in our case, probably indicative of mitotic entry induced by 17AAG treatment. Thus, although Myt1 is important in regulating the cyclin B cdc2 activity, it is unlikely to play a major role in abrogating the G2 M checkpoint by 17AAG. Wee1 Is an Hsp90 Client Protein in Mammalian Cells.
Chk1 has been implicated as an Hsp90 client protein that physically interacts with the molecular chaperone in whole cells based on coimmunoprecipitation studies. To demonstrate that Wee1 is also an Hsp90 client, cell lysate prepared from parental HCT116 cells were incubated with an Hsp90 specific or control IgG antibody. Endogenous Wee1 coimmunoprecipitated with Hsp90 only when an anti Hsp90 antibody was used. We next determined whether the depletion of Chk1 and Wee1 by 17AAG depends on the 26S proteasome. HCT116 parental cells were treated with 500 nM 17AAG in the presence or absence of the proteasome inhibitor MG 132 at three different concentrations. Coincubation with 17AAG and MG 132 resulted in near complete restoration of Chk1 protein level.
Down regulation of Wee1 by 17AAG was partially protected by cotreatment with MG 132, suggesting the possibility of a proteasome independent degradative process. To examine the effect of Hsp90 inhibition on Wee1 protein stability more directly, we performed a methionine labeled pulse chase experiment in control or 17AAG treated HCT116 cells. After a 30 min pulse with methionine, the level of radiolabeled Wee1 was followed during a 6 h chase period. In untreated cells, the half life of newly synthesized Wee1 was estimated to be 3.5 h. In the presence of 500 nM 17AAG, the half life of Wee1 was shortened to 1.6 h. It is noteworthy that the level of radiolabeled Wee1 at the beginning of the chase was not affected by 17AAG treatment, indicating that Hsp90 inhibition did not affect the translation of Wee1. To rule out an effect of Hsp90 inhibition on mRNA expression, we compared the abundance of Wee1 message in HCT116 cells treated sequentially with SN 38 followed by either dr