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During asymmetric cell division, cell polarity and cell cycle progression are tightly coordinated, yet mechanisms controlling both these events are poorly understood. Here we show that the Bora homologue SPAT-1 regulates both PAR polarity and cell cycle progression in C. elegans embryos. We find that, similarly to mammalian cells, SPAT-1 acts with PLK-1 and not with the mitotic kinase Aurora A (AIR-1), as shown in Drosophila. SPAT-1 binds to PLK-1, and depletion of SPAT-1 or PLK-1 leads to similar cell division defects in early embryos, which differ from the defects caused by depletion of AIR-1. Additionally, SPAT-1 and PLK-1 depletion causes impaired polarity with abnormal length of the anterior and posterior PAR domains, and partial plk-1(RNAi) or spat-1(RNAi), but not air-1(RNAi), can rescue the lethality of a par-2 mutant. SPAT-1 is enriched in posterior cells, and this enrichment depends on PAR polarity and PLK-1. Taken together, our data suggest a model in which SPAT-1 promotes the activity of PLK-1 to regulate both cell polarity and cell cycle timing during asymmetric cell division, providing a link between these two processes.

BackgroundRadiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions.Principal findingsHere we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-beta type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation.ConclusionsThese results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1GTP, which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1GDP, caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.

The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.

We have previously shown that histone H3 is transiently phosphorylated at Thr3 during mitosis. Extending these studies, we now report that phosphorylated Thr3 is always in cis to trimethylated Lys4 and dimethylated Arg8, forming a new type of combinatorial modification, which we have termed PMM. PMM-marked chromatin emerges at multiple, peripheral sites of the prophase nucleus, then forms distinct clusters at the centric regions of metaphase chromosomes, and finally spreads (as it wanes) to the distal areas of segregating chromatids. The characteristic prophase pattern can be reproduced by expressing ectopically the kinase haspin at interphase, suggesting that the formation of the PMM signature does not require a pre-existing mitotic environment. On the other hand, the ;dissolution' and displacement of PMM clusters from a centric to distal position can be induced by partial dephosphorylation or chromosome unravelling, indicating that these changes reflect the regulated grouping and scrambling of PMM subdomains during cell division. Formation of PMM is prevented by haspin knockdown and leads to delayed exit from mitosis. However, PMM-negative cells do not exhibit major chromosomal defects, suggesting that the local structures formed by PMM chromatin may serve as a ;licensing system' that allows quick clearance through the metaphase-anaphase checkpoint. J Cell Sci

Cul3 (Cullin3)-based E3 ubiquitin ligases recently emerged as critical regulators of mitosis. In this study, we identify two mammalian BTB (Bric-a-brac–Tramtrack–Broad complex)-Kelch proteins, KLHL21 and KLHL22, that interact with Cul3 and are required for efficient chromosome alignment. Interestingly, KLHL21 but not KLHL22 is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, similar to the previously described BTB-Kelch proteins KLHL9 and KLHL13. KLHL21 directly binds to Aurora B and mediates ubiquitination of Aurora B in vitro. In contrast to KLHL9 and KLHL13, KLHL21 localizes to midzone microtubules in anaphase and recruits Aurora B and Cul3 to this region. Together, our results suggest that different Cul3 adaptors nonredundantly regulate Aurora B during mitosis, possibly by ubiquitinating different pools of Aurora B at distinct subcellular localizations., The Journal of Cell Biology, 187 (6), ISSN:0021-9525, ISSN:1540-8140

AIM: We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. METHODS: We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). RESULTS: Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. CONCLUSION: Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing.

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Domains rich in alternating arginine and serine residues (RS domains) are found in a large number of eukaryotic proteins involved in several cellular processes. According to the prevailing view RS domains function as protein interaction domains, thereby promoting the assembly of higher-order cellular structures. Furthermore, recent data demonstrated that the RS regions of several SR splicing factors directly contact the pre-mRNA in a nonsequence specific but functionally important fashion. Using a variety of biochemical approaches, we now demonstrate that the RS domains of three proteins, not directly associated with the splicing reaction, such as lamin b receptor, acinus and peroxisome proliferator-activated receptor gamma coactivator-1 alpha, associate mainly with nuclear RNA and that this association is conducive in retaining the proteins in a soluble form. Phosphorylation by SRPK1 prevents RNA association, yet it greatly increases the fraction of the proteins recovered in soluble form, thereby mimicking the RNA effect. Based on these results we propose that the tendency to self-associate and form aggregates is a general property of RS domain-containing proteins and could be attributed to their disordered structure. RNA binding or SRPK1-mediated phosphorylation prevents aggregation and may serve to modulate the RS domain interaction modes. Biochim Biophys Acta

Dietary nutrient limitation (dietary restriction) is known to increase lifespan in a variety of organisms. Although the molecular events that couple dietary restriction to increased lifespan are not clear, studies of the model eukaryote Saccharomyces cerevisiae have implicated several nutrient-sensitive kinases, including the target of rapamycin complex 1 (TORC1), Sch9, protein kinase A (PKA) and Rim15. We have recently demonstrated that TORC1 activates Sch9 by direct phosphorylation. We now show that Sch9 inhibits Rim15 also by direct phosphorylation. Treatment of yeast cells with the specific TORC1 inhibitor rapamycin or caffeine releases Rim15 from TORC1-Sch9-mediated inhibition and consequently increases lifespan. This kinase cascade appears to have been evolutionarily conserved, suggesting that caffeine may extend lifespan in other eukaryotes, including man.

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Gα regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP2 synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Gα regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP2. We propose that asymmetric generation of PIP2 by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement., Developmental Cell, 15 (2), ISSN:1534-5807, ISSN:1878-1551

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.

Hypertension and hyperlipidemia, two powerful risk factors of cardiovascular disease (CVD), often coexist. Therefore, treatment should consider the beneficial properties of drugs used to treat either condition. Statins, the mainstay of lipid-lowering therapy, result in a significant clinical benefit both in primary and secondary CVD prevention. In addition to their hypolipidemic capacity, other properties may contribute to statin-induced benefits. Clinical and experimental evidence indicates that statins may modulate blood pressure (BP). The mechanisms by which statins reduce BP seem to be largely independent of their lipid effects. Although small, reductions in BP are possibly clinically relevant. Large landmark studies confirm that statins can reduce CVD risk in hypertensive patients. These findings suggest that statins could be prescribed as an adjunct in treating hypertension with dyslipidemia or even in patients with "normal" cholesterol levels. Whether the effect of statins on BP is accompanied by an additional decrease in clinical outcomes needs to be investigated in long-term, large-scale trials. Curr Hypertens Rep

The cell cycle-regulated Aurora-B kinase is a chromosomal passenger protein that is implicated in fundamental mitotic events, including chromosome alignment and segregation and spindle checkpoint function. Aurora-B phosphorylates serine 10 of histone H3, a function that has been associated with mitotic chromatin condensation. We find that activation of poly(ADP-ribose) polymerase (PARP) 1 by DNA damage results in a rapid block of H3 phosphorylation. PARP-1 is a NAD + -dependent enzyme that plays a multifunctional role in DNA damage detection and repair and maintenance of genomic stability. Here, we show that Aurora-B physically and specifically associates with the BRCT (BRCA-1 C-terminal) domain of PARP-1. Aurora-B becomes highly poly(ADP-ribosyl)ated in response to DNA damage, a modification that leads to a striking inhibition of its kinase activity. The highly similar Aurora-A kinase is not regulated by PARP-1. We propose that the specific inhibition of Aurora-B kinase activity by PARP-1 contributes to the physiological response to DNA damage.

Germline mutations of the LKB1 (STK11) tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) and predisposition to cancer. LKB1 encodes a serine/threonine kinase generally inactivated in PJS patients. We identified the dual phosphatase and tumor suppressor protein PTEN as an LKB1-interacting protein. Several LKB1 point mutations associated with PJS disrupt the interaction with PTEN suggesting that the loss of this interaction might contribute to PJS. Although PTEN and LKB1 are predominantly cytoplasmic and nuclear, respectively, their interaction leads to a cytoplasmic relocalization of LKB1. In addition, we show that PTEN is a substrate of the kinase LKB1 in vitro. As PTEN is a dual phosphatase mutated in autosomal inherited disorders with phenotypes similar to those of PJS (Bannayan-Riley-Ruvalcaba syndrome and Cowden disease), our study suggests a functional link between the proteins involved in different hamartomatous polyposis syndromes and emphasizes the central role played by LKB1 as a tumor suppressor in the small intestine.

The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350-403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/threonine kinases.

Summary Background SAK/PLK4 is a distinct member of the polo-like kinase family. SAK −/− mice die during embryogenesis, whereas SAK +/− mice develop liver and lung tumors and SAK +/− MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. Results Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. Conclusions SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outside-in" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the beta3 integrin, thereby amplifying outside-in signaling via alphaIIbbeta3. Blocking the Gas6-R-alphaIIbbeta3 integrin cross-talk might be a novel approach to the reduction of thrombosis.

Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P(2)-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P(2)-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P(2) during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P(2) disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P(2) metabolism regulates the dynamics and the function of Phg2.Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P(2)-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P(2)-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P(2) during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P(2) disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P(2) metabolism regulates the dynamics and the function of Phg2.

We have shown previously that culture of beta-cells on matrix derived from 804G cells and rich in laminin-5 improves their function. The purpose of this study was to investigate whether this matrix protects beta-cells against apoptosis and to elucidate signaling pathways involved. Matrix protected sorted rat beta-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1beta (IL-1beta; 2 ng/ml), compared with control (poly-L-lysine [pLL]). Caspase-8 activity was reduced in cells cultured on matrix, whereas focal adhesion kinase (FAK), protein kinase B (PKB, or Akt), and extracellular signal-regulated kinase (ERK) phosphorylation was augmented. Treatment (4 h) with an anti-beta1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on 804G matrix but not on pLL. In long-term assays (48 h), PD98059 but not LY294002 drastically augmented cell death on 804G matrix but did so to a lesser extent on pLL. The protein inhibitor of nuclear factor-kappaB (IkappaBalpha) was overexpressed in cells cultured 18 h on matrix with partial blockade by PD98059. In summary, this study provides evidence for activation of signaling pathways and gene expression by extracellular matrix leading to improved beta-cell survival.

Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation., In fission yeast, cell growth is co-ordinated with division by the cell tip-localized DYRK kinase Pom1, which inhibits the medially placed mitotic activator Cdr2. Here, Kelkar and Martin show that, upon glucose starvation, microtubules are destabilized in a PKA-dependent manner, leading to the deposition of Pom1 at cell sides where it delays mitosis.

NC2 is a heterodimeric regulator of transcription that plays both positive and negative roles in vivo. Here we show that the alpha and beta subunits of yeast NC2 are not always associated in a tight complex. Rather, their association is regulated, in particular by glucose depletion. Indeed, stable NC2 alpha/beta complexes can only be purified from cells after the diauxic shift when glucose has been depleted from the growth medium. In vivo, the presence of NC2 alpha, but not NC2 beta, at promoters generally correlates with the presence of TBP and transcriptional activity. In contrast, increased presence of NC2 beta relative to TBP correlates with transcriptional repression. NC2 is regulated by phosphorylation. We found that mutation of genes encoding casein kinase II (CKII) subunits as well as potential CKII phosphorylation sites in NC2 alpha and beta affected gene repression. Interestingly, NC2-dependent repression in the phosphorylation site mutants was only perturbed in high glucose when NC2 beta and NC2 alpha are not associated, but not after the diauxic shift when NC2 alpha and beta form stable complexes. Thus, the separation of NC2 alpha and beta function indicated by these mutants also supports the existence of multiple NC2 complexes with different functions in transcription.