4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) was identified as anThe pyrazolo-pyrimidine compound PP11 was identified as a high potency inhibitor of Src tyrosine kinase family members that acts as a competitive inhibitor of ATP binding (1). PP1 has been used in a number of studies to evaluate the role of Src tyrosine kinases in cellular function (2-7). PP1 does not affect the activity of other non-receptor tyrosine kinases, such as Jak-2 and Zap-70 (1). Src kinases have been reported to be critically involved in signaling via the stem cell factor (SCF) receptor c-Kit; inhibition of Src kinases has been shown to abrogate SCF induced proliferation of hemopoietic (3) and small cell lung cancer cells (8) and to block internalization of c-Kit after ligand binding (4). These findings have relied largely on the use of PP1 as a Src-selective inhibitor. c-Kit is expressed on hemopoietic progenitors, mast cells, and germ cells (for review, see Refs. 9 and 10). Loss of either c-Kit or its ligand in mice results in impaired hemopoiesis (in particular anemia), abnormalities of mast cell development, and impaired melanogenesis and gametogenesis (11). c-Kit has been implicated in tumor cell development, including mast cell disorders, (12) acute myeloid leukemia, gastrointestinal stromal tumors (13), Ewing's sarcoma, peripheral neuro-ectodermal tumors, malignant melanoma (11) and cancers of lung, ovary, and breast (14 -16). In some instances, this is because of activating mutations in c-Kit (e.g. in mastocytosis, acute myeloid leukemia, and gastrointestinal stromal tumors) (17); in others, the receptor is wild type but there is associated autocrine production of SCF (16).The development of inhibitors of c-Kit-mediated signals would be useful to investigate the role of c-Kit in maintaining these tumors and could provide the basis for a potential therapeutic agent. In this study, we have examined the effects of PP1 on signaling via c-Kit. We find that PP1 and the related compound PP2 block SCF-induced proliferation and activation of the ERK mitogen-activated protein kinase and Akt. This is associated with an inhibition of c-Kit autophosphorylation both in intact cells and in in vitro kinase assays. PP1 decreases the activity of constitutively active c-Kit and triggers the apoptosis of mast cell leukemia cells expressing this mutant. PP1 also inhibits Bcr-Abl kinase activity and triggers apoptosis in BcrAbl-dependent cells. These results have implications for the use of PP1 and related compounds in the experimental investigation of cell signaling pathways and suggest that this family of molecules may be useful in the development of treatment for diseases with abnormal c-Kit signaling. MATERIALS AND METHODSCell Culture-The RBL-2H3 cell line (18) (kindly supplied by S. Cockcroft,

To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.

Summary Anaphase-promoting complex or cyclosome (APC/C) controls the metaphase-to-anaphase transition and mitosis exit by triggering the degradation of key cell cycle regulators such as securin and B-type cyclins. However, little is known about the functions of individual APC/C subunits and how they might regulate APC/C activity in space and time. Here, we report that two potential Cdk1 kinase phosphorylation sites are required for the chromosomal localisation of GFP::Cdc27 during mitosis. Either or both of the highly conserved proline residues in the Cdk1 phosphorylation consensus sequence motifs were mutated to alanine (Cdc27 P304A or P456A). The singly mutated fusion proteins, GFP::Cdc27P304A and GFP::Cdc27P456A, can still localise to mitotic chromosomes in a manner identical to wild-type GFP::Cdc27 and are functional in that they can rescue the phenotype of the cdc27L7123 mutant in vivo. However, when both of the Cdk1 phosphorylation sequence motifs were mutated, the resulting GFP::Cdc27P304A,P456A construct was not localised to the chromosomes during mitosis and was no longer functional, as it failed to rescue mutant phenotypes of the cdc27L7123 gene. High levels of cyclin B and cyclin A were detected in mutant third instar larvae brain samples compared with its wild-type control. These results show for the first time that the two potential Cdk1 phosphorylation sites on Drosophila Cdc27 are required for its chromosomal localisation during mitosis and imply that these localisations specific to Cdc27 are crucial for APC/C functions.