We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.

Rapamycin is an immunosuppressant which antagonizes cellular proliferation by inhibiting the function of mTOR. The mTOR : FKBP12 : rapamycin complex blocks G1/S transition by inhibiting downstream targets essential for cell cycle progression. One such target is p70 S6k1 (S6K1), a serine/threonine kinase which is inactivated by the mTOR : FKBP12 : rapamycin complex, and which has been linked to translational control by virtue of its ability to phosphorylate the ribosomal protein S6. In the current work, we describe cloning and characterization of a novel S6K1 homolog, p54 S6 kinase 2 (p54 S6k2 /S6K2). Similar to S6K1, S6K2 is activated by mitogens and by constitutively active PI3K, and is inhibited by rapamycin as well as wortmannin. Dierences between activation of S6K1 and S6K2 by PDK1 were observed, suggesting potential dierences in the regulation of these homologs. Strikingly, S6K2 activity and S6 phosphorylation were both intact in S6K1 7/7 ES cell, indicating a possible role for S6K2 in in vivo S6 phosphorylation. Interestingly, we found two isoforms of S6K2 which are localized to distinct cellular compartments; the smaller form resides in the detergentsoluble fraction, whereas the larger form is found in the particulate fraction. Our ®ndings demonstrate the existence of a family of rapamycin-sensitive protein kinases potentially involved in S6 phosphorylation, translational control, and transduction of mTOR signals.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin þ perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.