Supplemental material for Hernandez et al. Dicoumarol downregulates human PTTG1/Securin mrna expression. through inhibition of Hsp90

Transcription

1 Supplemental material for Hernandez et al. Dicoumarol downregulates human PTTG1/Securin mrna expression through inhibition of Hsp90 Dicoumarol-Sepharose co-precipitation. Hsp90 inhibitors can co-precipitate Hsp90 polypeptides from cell extracts when these compounds are covalently linked to sepharose beads. As a complementary experiment to yeast sensitation to dicoumarol by overexpression of human Hsp90β and yeast Hsp82, we did pull-down experiments to ascertain if the less sensitive yeast Hsp82, and therefore probably the human orthologue, physically interacted with dicoumarol. For this, we bound dicoumarol to epoxy-activated sepharose beads and mixed them with yeast cell extracts. After extensive washing, co-precipitation experiments showed Hsp82 polypeptides on Western blots (Figure S1), indicative of a physical interaction between dicoumarol and this chaperone. Akt and regulation of Securin protein levels. It has been reported by two independent groups that PTTG1/Securin expression is dependent on the IGF-I/PI3K/Akt pathway (1, 2). This is in clear contrast to our own results (main text). For this reason, we tested if wortmannin, an inhibitor of PI3K, altered Securin protein amounts. Indeed, wortmannin (10 µm) reduced drastically the levels of Securin in HCT116 and Hela cells (Fig S2a and data not shown). We tested then if Akt mutants altered the wortmannininduced drop in Securin levels. Shockingly, overexpression of either mutant had no effect on Securin protein amounts (Figure S2a). Wortmannin has off-target effects at high concentrations (3) while it is fairly specific for class I PI3K in the

2 nanomolar range. Therefore, we did a dose-response assay to test if reduction of Securin amounts and death induction was concomitant to PI3K inhibition. To this end, we probed the levels of Ser473-phosphorylated Akt as a marker for PI3K activity, along with levels of Securin, PARP-1 and the active 18 kda fragment of caspase 3. Phosphorylation of Akt dropped at concentrations above 250 nm while the onset of death induction and Securin downregulation was observed when wortmannin was added at and above 5 µm in the medium (Figure S2b). We finally tested if Securin protein levels were sensitive to IGF-I in our cell line. We starved cells in serum-free medium for 24 hours prior to addition of IGF-I and wortmannin. Twenty-four hours later, Securin protein levels were assessed by western blot. Figure S2c shows that, after serum starvation for 48 hours, Securin levels were merely ca 2-fold lower than those from non-starved controls. Strikingly, addition of 100 ng/ml IGF-I to serumstarved cells did not produce an increase in Securin levels. Moreover, 0.5 µm wortmannin (a concentration inducing PI3K inhibition, as seen on Figure S2b) had no effect on Securin levels. Similarly, addition of PD (50 µm) to IGF-I supplemented cells had no effect on Securin levels (lane marke as Ε in Figure S2c) Our data show clearly that Akt inhibiton is not the cause for Securin mrna repression in cervix (Hela) and colorectal cancer cells (HCT116), although Akt is clearly affected by dicoumarol and the IGF-I/PI3K/Akt pathway being involved in PTTG1/Securin regulation in normal and some tumour cells. This discrepancy is explained by Securin expression in our cell lines being, for the most part, impervious to serum deprivation and insensitive to the addition of IGF-I. Independence from serum growth factors is a common trait in tumour cells.

3 Methods Epoxy-activated sepharose beads (Sigma-Aldrich) were swollen in distilled water, washed and coupled to dicoumarol by overnight incubation in 50 mm NaOH/KCl buffer ph 14 at 4 o C. After washing uncoupled dicoumarol, remaining free epoxy groups were blocked with 1 M β-mercaptoethanol for 4 h at room temperature in NaOH/KCl buffer and 4 later washes at alternating acid and alkaline ph. Mock beads were obtained in the absence of dicoumarol in the coupling buffer. Yeast expressing phsp82-flag were grown to early logarithmic phase and total cell extracts obtained by breaking cells with glass beads (0.5 mm diameter) in YB buffer (50 mm Tris/HCl ph 7.5, 10% glycerol, 5 mm dithiotreitol, 2.5 mm EDTA, 1 mm benzamidine, 1 mm PMSF and protease inhibitors (Roche)). Cell extracts were clarified by centrifugation at 20,000xg for 15 min. Cell extracts (100 µg protein) were mixed with 30 µl of dicoumarolsepharose beads in 650 µl of YB buffer. After overnight incubation at 4 o C with rocking, beads were extensively washed with YB buffer. Attached proteins were solubilised from dry beads with Laemli loading buffer and subjected to Western blotting using anti-flag antibodies (Sigma-Aldrich), as above. Plasmids carrying wild-type, myristoylatable or dominant negative mutant forms of Akt/PKB are described in (4, 5). Transfections were done as described in the main text.

4 References 1. Thompson AD, 3rd, Kakar SS. Insulin and IGF-1 regulate the expression of the pituitary tumor transforming gene (PTTG) in breast tumor cells. FEBS Lett 2005;579: Chamaon K, Kirches E, Kanakis D, Braeuninger S, Dietzmann K, Mawrin C. Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-i and insulin differs between malignant and non-neoplastic astrocytes. Biochem Biophys Res Commun 2005;331: Knight ZA, Shokat KM. Features of selective kinase inhibitors. Chem Biol 2005;12: Salinas M, Wang J, Rosa de Sagarra M, et al. Protein kinase Akt/PKB phosphorylates heme oxygenase-1 in vitro and in vivo. FEBS Lett 2004;578: Salinas M, Lopez-Valdaliso R, Martin D, Alvarez A, Cuadrado A. Inhibition of PKB/Akt1 by C2-ceramide involves activation of ceramide-activated protein phosphatase in PC12 cells. Mol Cell Neurosci 2000;15:

5 Dicoumarol beads Figure S1 Input (1/10) Mock beads

6 Figure S2 A pcdna3 pakt (myr) pakt (K179M) Wortmannin Akt Securin β-actin B Wortmannin (µm) PARP-1 Caspase 3, 18 kda fragment Securin Akt Akt (Ser473-p) β-actin C Ε - + Serum IGF-I Wortmannin Securin β-actin