In addition, sequence analysis indicated that Ser112 and Ser394 are a part of Ser-Pro sequences, which can be phosphorylated by Cdks or the mitogen-activated protein kinase family. down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis exhibited that Ser21 phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is usually phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity and conversation with DNA in cells. Human topo I2 is usually a type IB topoisomerase that relieves positive and negative DNA supercoiling caused by transcription, replication, and chromosome condensation (1). The 91-kDa, 765-amino acid polypeptide contains four domains: a poorly conserved lysine-rich N-terminal domain name that contains nuclear and nucleolar localization signals, Cariprazine hydrochloride a linker region, and the core and C-terminal domains that contain the residues important for DNA conversation and relaxation of supercoils (2). A transesterification reaction at the active site of topo I ligates Tyr723 of the enzyme to the 3 phosphate of the DNA, thereby creating a nick in the DNA backbone (3). This nick allows controlled rotation of the DNA to relieve supercoils. Mutation of Tyr723 prevents the transesterification reaction and abolishes all relaxation activity (4). The anticancer drug CPT and its derivatives slow topo I-mediated DNA relaxation (5, 6) and inhibit the religation reaction step of the enzyme (7, 8), trapping topo I on DNA (9, 10) and causing cell death (11, 12). A number of observations have raised the possibility that phosphorylation can modulate the Cariprazine hydrochloride activity and CPT sensitivity of topo I. Treatment with calf intestine alkaline phosphatase decreases topo I enzymatic activity or and in intact cells. In addition, we performed site-directed mutagenesis of these sites to assess the impact on localization and activity. Results of this analysis suggest that topo I is usually phosphorylated during mitosis in untreated cells and that one of these mitotic phosphorylations modestly enhances topo I activity as well as sensitivity to CPT-induced trapping on DNA in intact cells. EXPERIMENTAL PROCEDURES kinase assays as described below. S-Topo I was used for all other experiments. for 15 min. Topo I antibody was added to the resulting supernatant, which was rotated end over end overnight. Samples were supplemented with protein A beads and rotated for an additional 4 h. Beads were spun down at 12,000 for 1 min and washed four occasions with RIPA Cariprazine hydrochloride buffer made up of phosphatase inhibitors (1% (w/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 150 mm NaCl, 10 mm sodium phosphate (pH 7.2), 2 mm EDTA, 1 mm sodium orthovanadate, 100 models/ml Trasylol, 50 mm NaF). To elute protein for SDS-PAGE, beads were resuspended in sample buffer (4 m urea, 2% (w/v) SDS, 62.5 mm Tris-HCl (pH 6.8), 1 mm EDTA, 5% (v/v) 2-mercaptoethanol, 0.1% (w/v) bromphenol blue) and heated to 65 C for 20 min. S-Topo I or Topo I-S was isolated using a comparable procedure. In brief, lysates were prepared as described above and sedimented at 12,000 for 15 min. S protein beads (Novagen) were added to the resulting supernatant, which was rotated end over end overnight. Beads were then sedimented and washed four occasions with RIPA buffer made up of phosphatase inhibitors prior to SDS-PAGE or incubation with kinases. Alternatively immobilized topo I was washed with 0.1% Nonidet P-40 in PBS rather than RIPA buffer and assayed for topo I activity. (27). The resulting sample was spotted onto a 20 20-cm, 100-m cellulose thin layer chromatography plate (EM Science), subjected TSPAN11 to electrophoresis at 1000 V for 30 min in pH 1.9 buffer, and exposed to ascending chromatography overnight in buffer consisting of 37.5% (v/v) for 5 Cariprazine hydrochloride min using a Shandon cytocentrifuge. Cells were fixed in ice-cold methanol for 10 min, rehydrated in PBS, and blocked in TSM (150 mm NaCl, 10 mm Tris-HCl (pH 7.4) containing 10% (w/v) nonfat milk powder, 100 models/ml penicillin G, 100 g/ml streptomycin, 1 mm sodium azide) at 20C22 C for 1 h. After anti-S.