Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.

In higher eukaryotes, Cdk2 kinase plays an essential role in regulating the G1-S transition. Here, we use cycling Xenopus egg extracts to examine the requirement of Cdk2 kinase on progression into mitosis. Interestingly, when Cdk2 kinase activity is inhibited by the Cdk-specific inhibitor, p21Cip1, a block to mitosis occurs, and inactive Cdc2-cyclin B accumulates. This block occurs in the absence of nuclei and is not due to direct inhibition of Cdc2 by Cip. Importantly, this block to mitosis is reversible by restoring Cdk2-cyclin E kinase activity to a Cip-treated cycling extract. Moreover, immunodepletion of Cdk2 from interphase extracts prevents activation of Cdc2 upon the addition of exogenous cyclin B. Thus, our data show that Cdk2 kinase is a positive regulator of Cdc2-cyclin B complexes and establish a link between Cdk2 kinase and cell cycle progression into mitosis.

Abstract. We have developed methodology to identify the block to anchorage-independent growth and position it within the fibroblast cell cycle . Results with NRK fibroblasts show that mitogen stimulation of the GO/G1 transition and GI-associated increases in cell size are minimally affected by loss of cell anchorage . In contrast, the induction of Gl/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked . Moreover, we demonstrate that the anchorage-dependent transition maps to late Gl and shortly before activation of the Gl/S p34cdc 2 -like kinase .

The p42 mitogen-activated protein kinase (MAPK) is required for progression through meiotic M phase in Xenopus oocytes. This report examines whether it also plays a role in normal mitotic progression. MAPK was transiently activated during mitosis in cycling Xenopus egg extracts after activation of the cyclin-dependent kinase Cdc2-cyclin B. Interference with MAPK activation by immunodepletion of its activator MEK, or by addition of the MEK inhibitor PD98059, caused precocious termination of mitosis and interfered with production of normal mitotic microtubules. Sustained activation of MAPK arrested extracts in mitosis in the absence of active Cdc2-cyclin B. These findings identify a role for MEK and MAPK in maintaining the mitotic state.

Aberrant cell division is a hallmark of cancer, but the molecular circuitries of this process in tumor cells are not well understood. Here, we used a high-throughput proteomics screening to identify novel molecular partners of survivin, an essential regulator of mitosis overexpressed in cancer. We found that survivin associates with the small GTPase Ran in an evolutionarily conserved recognition in mammalian cells and Xenopus laevis extracts. This interaction is regulated during the cell cycle, involves Ran-GTP, requires a discrete binding interface centered on Glu65 in survivin, and is independent of the Ran effector Crm1. Disruption of a survivin-Ran complex does not affect the assembly of survivin within the chromosomal passenger complex or its cytosolic accumulation, but it inhibits the delivery of the Ran effector molecule TPX2 to microtubules. In turn, this results in aberrant mitotic spindle formation and chromosome missegregation in tumor, but not normal, cells. Therefore, survivin is a novel effector of Ran signaling, and this pathway may be preferentially exploited for spindle assembly in tumor cells.Deregulated cell division is a hallmark of cancer (31), which results in unrestrained cell proliferation, abrogation of cell cycle checkpoints, and propensity to aneuploidy (27). These processes hinge on spatial-temporal assembly of a bipolar mitotic spindle (23), where microtubules nucleating from duplicated centrosomes or assembled in proximity of mitotic chromosomes "search and capture" chromatids and ultimately segregate them between daughter cells (46). Reconstitution experiments in model organisms, particularly cycled Xenopus laevis extracts, uncovered a pivotal role of the small GTPase Ran in the mechanism of spindle formation (36). We now know that a gradient of Ran-GTP assembles on mitotic chromosomes (20), largely through the chromatin-associated activity of the nucleotide exchange factor RCC1. In turn, this releases Ran effector molecules implicated in spindle formation (9, 15), including microtubule-stabilizing TPX2 (14), from an inhibitory interaction with importin ␣/␤ receptors (16, 45). Depletion of Ran in Xenopus extracts (9) or mammalian cells (15) or targeting its effector molecules (26, 41, 43) profoundly impairs spindle formation, causing the appearance of flattened mitotic spindles, severely depleted of microtubules, and abnormal chromosomal segregation.Among the regulators of cell division aberrantly overexpressed in cancer is survivin (3), a member of the inhibitor of apoptosis (IAP) gene family with dual roles in suppression of cell death and control of mitosis (1, 29). The latter has been linked to a multiplicity of functions, including kinetochore targeting of the chromosomal passenger complex (22), enhancement of Aurora B kinase activity (7), control of kinetochore-microtubule interactions for proper chromosomal alignment (39), participation in the spindle assembly checkpoint (11), and regulation of microtubule dynamics for spindle formation (37). Although these functions are ess...