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Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.entromere-associated protein-E (CENP-E; kinesin-7) is a kinetochore-associated kinesin motor protein with an essential and exclusive role in metaphase chromosome alignment and satisfaction of the mitotic checkpoint (1). CENP-E is a likely candidate to integrate the mechanics of kinetochore-microtubule interaction with the mitotic checkpoint signaling machinery responsible for restraining cell-cycle progression into anaphase. CENP-E is a large dimeric protein consisting of an N-terminal kinesin motor domain tethered to a globular C-terminal domain through an extended coiled-coil rod domain (2, 3). The C-terminal, noncatalytic region of CENP-E is not only sufficient to specify localization to kinetochores, but it also mediates interaction of CENP-E with the serine/threonine kinase BubR1, a key effector of mitotic checkpoint signaling that forms complexes with the checkpoint proteins Cdc20, Bub3, and Mad2 to inhibit the ubiquitin ligase activity of the anaphase promoting complex APC/C CDC20 (4-7). The combined interaction of CENP-E with microtubules and a key regulator of APC/C CDC20 has led to the hypothesis that CENP-E functions as the key kinetochore microtubule receptor responsible for silencing mitotic checkpoint signal transduction after capture of spindle microtubules. This hypothesis was further strengthened by the finding that CENP-E could stimulate the kinase activity of BubR1 in a microtubule-sensitive manner (8, 9). In vitro, the addition of CENP-E to BubR1 resulted in a stimulation of BubR1 kinase activity. The addition of microtubules suppressed this stimulatory activity, an effect thought to be mediated by the CENP-E kinesin motor domain. Although the importance of CENP-E interaction with BubR1 and the role of BubR1-mediated phosphorylation in mitotic checkpoint function remain unclear, CENP-E remains a prominent candidate to play a key role in mitotic checkpoint signal transduction.Depletion of CENP-E from ...

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytosplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBPI) Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target ofFKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.The macrolide drug rapamycin exhibits immunosuppressive as well as antineoplastic and antiproliferative properties (reviewed in reference 52). Despite the structural similarity between rapamycin and FK506, FK506 (as well as the cyclic undecapeptide cyclosporin A [CsA]) abrogates early events in T-cell activation by specifically blocking transcription of interleukin-2 (IL-2) (47, 70; reviewed in references 62 and 64), whereas rapamycin blocks subsequent lymphokine receptor-mediated processes (16,18).The blockade of T-cell signal transduction results from the interaction of these agents with specific intracellular receptors (or immunophilins). CsA binds to a class of proteins called cyclophilins (reviewed in reference 73), whereas the primary targets for both rapamycin and FK506 are the FKBPs (for FK506-binding proteins) (28,67,69). One FKBP subtype (FKBP12) has been purified from a variety of organisms and, like the cyclophilins, shown to be an enzyme with peptidyl-prolyl cis-trans isomerase (PPIase) activity (28,67). It is well established, however, that although ligand binding specifically inhibits enzymatic activity in vitro, this loss of function is not required for immunosuppression (6,24,29,30,37,45,74 interacting with other downstream cellular proteins. Thus, the immunophilins act as chaperones for these drugs, delivering them to another site of action in the cell.Both the cyclophilin-CsA and FKBP12-FK506 complexes bind to a specific protein phosphatase (calcineurin) which is hypothesized to control the activity of IL-2 gene-specific transcriptional activators (12, 24, 45, 55; reviewed in reference 63). In contrast, the downstream cellular targets for the rapamycin-sensitive signaling pathway have not been genetically characterized, although rapamycin has been shown recently to block the phosphorylation and activation of 70-kDa S6 (pp7OS6K) and p34cdc2 kinases in animal cells (8,11,51).Since rapamycin is a potent antifungal agent, we have used the power of yeast genetics to rapidly dissect the rapamycin-sensitive pathway, with the hope that a parallel pathway exists in mammalian cells. We and others previously identified and characterized the gene encoding a yeast homolog of human FKBP12 (hFKB12) (29,30,37,39,74). Deletion of this gene (which we call RBP1, for rapamycinbinding protein; also known as FPRI and FKB1 [30,37,74]) results in a recessive rapamycin-resistant phenotype, and expression of human FKBP12 in an rbpl deletion mutant restores rapamycin sensitivity (37).In this study, we hav...

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the response to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, -13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBPI (for rapamycin-binding protein). The RBPI protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.Agents that inhibit T-cell activation include cyclosporin A (CsA) (19) and the recently discovered macrolide FK506 (31, 36). CsA was originally discovered as an antifungal agent, and FK506 was identified as an inhibitor of interleukin-2 (IL-2) production (20). Despite the structural dissimilarity between these two immunosuppressive drugs, recent reports suggest that the targets for both agents, cyclophilin and FK506-binding protein (FKBP), respectively, are peptidylprolyl cis-trans isomerases (PPlases), enzymes that promote protein folding in vitro (12,15,34,35,37,40,41). Although the endogenous function of PPlases is not known, the fact that the immunosuppressive action of CsA and FK506 is linked to inhibition of PPIase activity suggests that they may be required in the regulation of intracellular signaling events leading to T-cell activation (8, 12, 37).Rapamycin, a macrolide antifungal agent with structural similarity to FK506 (32, 42), also exhibits immunosuppressive (3, 26, 38) as well as antineoplastic (9, 18) properties. Rapamycin and FK506 act as reciprocal antagonists in vivo (murine T cell activation [6]) and compete for binding to FKBP (15). Given these similarities, the mechanism of action of rapamycin remains enigmatic because, whereas FK506 (like CsA) acts to inhibit IL-2 transcription, rapamycin has no effe...

To establish well-characterized cellular reagents for the study of colon carcinoma, we have examined 19 human colorectal carcinoma cell lines with regard to morphology, ultrastructure, expression of tumor-associated antigens, proliferative capacity in vitro, anchorage-independent growth, oncogene expression, tumorigenicity and malignant potential. Cell lines examined were cultured under identical conditions, and in vitro and in vivo analyses were performed in parallel on replicate cultures. Three classes of colorectal cell lines were defined according to their tumorigenicity in nude mice. Class-1 lines formed rapidly progressing tumors in nearly all mice at an inoculum of 10(6) cells. Cell lines belonging to class-2 were less tumorigenic, producing tumors later and at a slower growth rate. Class-3 lines were non-tumorigenic under all experimental conditions tested. By Northern analysis, the oncogenes c-myc, H-ras, K-ras, N-ras, myb, fos and p53 were expressed in nearly all cell lines examined. In contrast, transcripts for abl, src and ros were not detected. The best in vitro predictor of tumorigenicity was colony formation in soft agar. There was no detectable correlation between tumorigenicity and metastatic potential, doubling time in vitro, production of tumor-associated markers, xenograft histology or expression of specific oncogenes.