Previous work from our laboratory showed that tumor cells exposed to an anticancer mitotic inhibitor, regosertib (ON 01910.Na), resulted in G2/M arrest and hyperphosphorylation of RanGAP1·SUMO1 (HRGS). These findings suggest that regosertib may act either as an inhibitor of a RanGAP1·SUMO1 phosphatase or stimulant of a new kinase (Cancer Res 2011; 71; 4968-76). We further studied the relationship between dephosphorylation and deSUMOylation of HRGS. In the present study we used DU145 prostate cancer cell line. After exposure to one microM regosertib for 24 h, cells were collected and subjected, in equal portions, to lysis in different lysis buffers composed of basic lysis buffer and varied in supplemented inhibitors. Basic lysis buffer contained 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton; 0.5% NP-40; 1 mM EGTA and 1 mM EDTA. Thus, prepared cell lysates were analyzed by Western blot for expression of phosphorylated and non-phosphorylated forms of RanGAP1, RanGAP1·SUMO1, PP1 and Cdc25C. Results: Roche protease inhibitor cocktail, PMSF (0.5 mM), NaF (1 mM) and Na3VO4 (1mM) as inhibitors added to the basic lysis buffer were unable to protect against loss of SUMO-group from RanGAP1·SUMO1 and, concomitantly, against de-phosphorylation of PP1 phosphatase at T-320. However, addition of N-ethylmaleimide (NEM, an inhibitor of deSUMOylation) as a single inhibitor supplement in the basic lysis buffer resulted in concentration-dependent (2.5 ∼ 40mM) inhibition of deSUMOylation of HRGS. Yet, even at lower NEM concentration RanGAP1, devoid of the SUMO-group, was still phosphorylated. This might be in correlation with our finding that even low concentration of NEM in the lysis buffer kept PP1 in phosphorylated state at T320 (which is inhibitory to PP1), preventing it from auto-dephosphorylation and re-activation. Also, neither regosertib (up to 100 microM) nor okadaic acid (OA, up to 1 microM), added to the basic lysis buffer as putative inhibitors, could prevent loss of SUMO group from HRGS and dephosphorylation of PP1 at T320. Regosertib in the lysis buffer did not prevent dephosphorylation of hyperphosphorylated Cdc25C (aka mitotic Cdc25C), while NEM and OA did (the latter implies that dephosphorylation was carried out by PP1 in the lysate). Conclusion: These data show that hyperphosphorylated RanGAP1·SUMO1 can be deSUMOylated, but remains phosphorylated. DeSUMOylation may be a prerequisite for RanGAP dephosphosphorylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3797. doi:1538-7445.AM2012-3797