Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
The tumor suppressor gene PTEN is commonly mutated or deleted in human cancers, leading to the phosphorylation and activation of the proto-oncogene protein kinase AKT (PKB). Phosphorylated AKT inactivates several transcription factors in the nucleus, but little is known about its regulation in this compartment. In Nature, Pandolfi and colleagues now show that the tumor suppressor promyelocytic leukemia (Pml) keeps nuclear Akt in check.
The authors found that the life expectancy of heterozygous double-knockout Pten+/- Pml+/- mice was markedly reduced compared to Pten+/- Pml+/+ mice; this is due to accelerated formation and progression of tumors in the colon and prostate. Interestingly, loss of Pml in these tissues increased the levels of nuclear phosphorylated Akt in Pten+/- mice.
To determine whether Pml plays a role in Akt signaling, Pandolfi and colleagues assessed the status of Akt in Pml-/- mouse embryonic fibroblasts (MEFs). Serum stimulation of Pml-null cells enhanced the nuclear accumulation of phosphorylated Akt compared to wild type MEFs. This effect was rescued by PML expression, indicating that Pml may regulate Akt activation and localization.
In agreement with these findings, the transcription factor FOXO3a, which is normally inactivated by Akt and exported from the nucleus, was predominantly found in the cytoplasm of Pml-/- cells after serum stimulation. Furthermore, the transcription of FOXO3a targets, such as the proapoptotic factor Bim and the DNA repair protein Gadd45, was decreased in the absence of Pml.
So, how does Pml regulate Akt activity? Treatment of Pml+/+ MEFs with okadaic acid, which inhibits the only known Akt-Thr 308 phosphatase PP2a, increases the levels of phosphorylated Akt to those found in Pml-/- cells, suggesting that Pml inactivates Akt through PP2a. Indeed, both A and C subunits of PP2a co-precipitate with Pml; moreover, PP2a, Pml and Akt were found to co-localize in nuclear bodies of wild type MEFs but not in Pml-/- nuclei.
In summary, this paper suggests that Pml inactivates nuclear Akt by increasing the interaction between phosphorylated Akt and PP2a in this cellular compartment, thereby preventing Akt-driven tumorigenesis. The results indicate that this function of Pml is especially important after Pten loss, and that tissues like prostate and colon are particularly sensitive to the levels of active Akt. Whether Pml is also involved in the recruitment of phosphorylated Akt to the nucleus remains to be investigated.
article Lloyd C Trotman, Andrea Alimonti, Pier Paolo Scaglioni, Jason A Koutcher, Carlos Cordon-Cardo & Pier Paolo Pandolfi Identification of a tumour suppressor network opposing nuclear Akt function Nature, 441, 523-527 doi:10.1038/nature04809 Full text | PDF | Subscribe to Nature
Although cells transduce environmental signals through a limited set of canonical signaling pathways, the pathways themselves are activated by a sizeable array of ligands. Combinations of these ligands can generate a vast number of inputs; for example, 20 different ligands might in principle elicit over a million possible unique output responses. The Alliance for Cellular Signaling (AfCS) set out to examine the complexity of these responses by analyzing the level of cross-talk between 22 receptor-specific ligands in RAW 264.7 macrophages. This study, published in Nature Cell Biology, reveals novel signaling circuits and provides insight into context-dependent signaling by providing an estimate of the level of regulatory cross-talk in this system.
Ranganathan and colleagues selected representative ligands of diverse signaling pathways and measured signaling parameters such as cAMP synthesis, protein phosphorylation and intracellular calcium mobilization, as well as signaling outputs such as cytokine secretion. To test the robustness of their approach the authors constructed a clustered matrix representing the input and output responses for all 22 single ligands. This single ligand matrix was largely consistent with published signaling mechanisms.
A similar matrix of all 231 pair-wise ligand combinations was then assembled. Where no cross-talk occurs, the expected output of two ligands is simply additive. An output higher than this would indicate cross-talk and synergy, possibly via direct interaction or feedback mechanisms. This screen yielded predicted cases of synergistic signaling as well as several novel interactions. For example, combinations of Ca2+-mobilizing and cAMP-inducing ligands led to synergistic increases in cAMP in conjunction with the synergistic inhibition of Ca2+ mobilization. Cells treated with ionomycin, to raise levels of Ca2+ or EGTA to chelate intracellular calcium, revealed that Ca2+ mobilization alone is insufficient to support synergistic signaling. Ligand-mediated cAMP production is specifically upregulated by receptor-mediated calcium mobilization, representing a novel Ca2+-cAMP signaling circuit.
All ligands diplayed non-additive, synergistic activatory or inhibitory responses in different pair-wise combinations, with the majority of the ligand pairs’ output falling into a relatively small number of clusters, possibly representing pathway convergence. For example, the screen showed that Toll like receptor (TLR) signaling is modulated by a cAMP-dependent process. This is in agreement with published information that Gαs signaling can attenuate inflammatory responses due to TLR activity.
This study shows how a relatively small number of ligands are able to regulate downstream interactions in a context-dependent manner. The emerging basic architecture of the signaling network for the RAW 264.7 macrophage cell line can be expected to provide an overview of signaling cross-talk in other mammalian systems.
Clare Garvey, Assistant Editor Signaling Gateway
article Madhusudan Natarajan, Keng-Mean Lin, Robert C. Hsueh, Paul C. Sternweis & Rama Ranganathan A global analysis of cross-talk in a mammalian cellular signalling network Nature Cell Biology, (14 May 2006); doi:10.1038/ncb1418 Full text | PDF | Subscribe to Nature Cell Biology
The use of recombinant leptin, a fat-derived hormone that regulates food intake and energy metabolism, as an antiobesity drug is hindered by the development of leptin resistance in both human and rodent obesity. Ciliary neurotrophic factor (CNTF), a cytokine with leptin-like antiobesogenic properties, has recently emerged as a potential alternative for controlling obesity-related diseases, but its mechanism of action remains unexplored. Now in Nature Medicine, Watt et al. show that CNTF is able to reduce the synthesis of complex lipids in skeletal muscle by activating AMPK (AMP-activated protein kinase), improving insulin sensitivity, even where leptin resistance is observed.
The authors show that, like leptin, CNTF activates AMPK in skeletal muscle leading to an increase in fatty-acid oxidation. Both gp130, a member of the CNTF receptor complex, and AMPK activation are required for CNTF-dependent palmitate oxidation. Leptin and CNTF also activate signal transducer and activator of transcription (STAT3) to regulate the expression of gluconeogenic genes. It has previously been shown that leptin resistance is partly due to an upregulation of suppressor of cytokine signaling 3 (SOCS3) which inhibits the leptin-dependent activation of STAT3. However, in this study, Watt et al. show that STAT3 activation is not required for CNTF-induced AMPK signaling.
Peripheral, but not intracerebroventricular, CNTF administration leads to AMPK activation and the upregulation of genes encoding proteins that enhance fatty acid oxidation in red gastrocnemius muscle, suggesting that the effects of CNTF are independent of signaling through the brain. Importantly, CNTF is able to induce AMPK phosphorylation and palmitate oxidation in mice lacking functional leptin receptors or those fed a high-fat diet. Indeed, treatment of obese mice with CNTF reduces body mass and increases both fatty acid oxidation and insulin-stimulated glucose uptake in skeletal muscle. Furthermore, CNTF restores insulin sensitivity in lipid-induced insulin-resistant cells, indicating that CNTF overcomes both insulin and leptin resistance.
The ability of CNTF to prevent lipid buildup and improve insulin action in obese mice, without signaling through the brain and without apparent toxicity, holds promise for recombinant CNTF in the treatment of obesity and type 2 diabetes.
article Matthew J Watt, Nicolas Dzamko, Walter G Thomas, Stefan Rose–John, Matthias Ernst, David Carling, Bruce E Kemp, Mark A Febbraio & Gregory R Steinberg CNTF reverses obesity–induced insulin resistance by activating skeletal muscle AMPK Nature Medicine, 12, 541 – 548 doi:10.1038/nm1383 Full text |PDF | Subscribe to Nature Medicine
news and views Rexford S Ahima Overcoming insulin resistance with CNTF Nature Medicine, 12, 511 – 512 doi:10.1038/nm0506-511 Full text |PDF | Subscribe to Nature Medicine
Shugoshin: guardian of centromeric cohesin
In mitosis and meiosis, sister chromatids are bound together by the ring shaped cohesin complex. The centromeres of chromosomes assemble kinetochore complexes that mediate interaction with spindle microtubules. While cohesins detach from chromosome arms early in mitosis, cohesin around the centromere is dissolved much later, in anaphase, through cohesin cleavage by the protease separase. The persistence of centromeric cohesin requires a family of kinetochore localized proteins called the shugoshins. Two studies by Riedel et al. and Kitajima et al. in Nature now show that shugoshin (Sgo) recruits a subtype of protein phosphatase 2A (PP2A) to the centrosome, protecting cohesin from degradation. A further study by Brar et al. investigates whether phosphorylation of the meiosis specific cohesin subunit regulates the loss of centromeric cohesins.
Riedel et al. and Kitajima et al. both show that PP2A co-purifies with Sgo during meiosis in S. cerevisiae, S. pombe and human HeLa cells. PP2A is a hetero-trimeric complex, comprising A (scaffold), B (regulatory) and C (catalytic) subunits. Sgo interacts with PP2A that contains B’ type regulatory subunits. Indeed, B’ subunits are predominantly localized to the centrosome with Sgo proteins, while other PP2A subunits are localized throughout the cell. Reduction of PP2A protein levels in either yeast or HeLa cells increases the frequency of random chromatid segregation. Kitajima et al. show that the human cohesin subunit SA2 is dephosphorylated when chromatin-bound, while free SA2 remains phosphorylated. Furthermore, purified human Sgo can counteract the phosphorylation of SA2. Both groups reveal how tethering of PP2A to chromosomes prevents the removal of cohesins and leads to their dephosphorylation.Taken together, these results suggest that the main function of shugoshin is to recruit PP2A to the centrosome, where it dephosphorylates cohesin allowing for timely chromatid segregation.
A study by Brar et al. published online in Nature examines whether phosphorylation of cohesin contributes to cohesin loss at the centrosome during meiosis, following on from data showing that the Polo-like kinase Cdc5 is required for cleavage of the cohesin subunit Rec8 and consequently chromosome segregation. The authors define the putative sites of cohesin phosphorylation and mutate them. Only mutation of multiple sites delayed prophase of meiosis I. Surprisingly, Brar et al. show that deletion Spo11, a gene required for the creation of double stranded breaks and therefore meiotic recombination, abolishes the requirement for cohesin phosphorylation. Thus, Rec8 phosphorylation is important largely for the first meiotic division, while recombination in the second meiotic division is essential for silencing of spindle checkpoints and the timely removal of cohesins.
These studies reveal how Sgo recruits PP2A to the centrosome, so as to reduce the phosphorylation, cleavage and removal of cohesin. Cohesin phosphorylation is less important for its removal in the second meiotic cleavage where recombination occurs.
Clare Garvey, Assistant Editor Signaling Gateway
article Tomoya S Kitajima, Takeshi Sakuno, Kei-ichiro Ishiguro, Shun-ichiro Iemura, Tohru Natsume, Shigehiro A Kawashima & Yoshinori Watanabe Shugoshin collaborates with protein phosphatase 2A to protect cohesin Nature, 441, 46-52 doi:10.1038/nature04663 Full text | PDF | Subscribe to Nature
article Christian G Riedel, Vittorio L Katis, Yuki Katou Yuki Katou, Saori Mori, Takehiko Itoh, Wolfgang Helmhart, Marta Gálová, Mark Petronczki, Juraj Gregan, Bulent Cetin, Ingrid Mudrak, Egon Ogris, Karl Mechtler, Laurence Pelletier, Frank Buchholz, Katsuhiko Shirahige & Kim Nasmyth Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I Nature, 441, 53-61 doi:10.1038/nature04664 Full text | PDF | Subscribe to Nature
news and views Paul Megee Molecular biology: Chromosome guardians on duty Nature, 441, 35-37 doi:10.1038/441035a Full text | PDF | Subscribe to Nature
letter Gloria A. Brar, Brendan M. Kiburz, Yi Zhang, Ji-Eun Kim, Forest White and Angelika Amon Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis Nature, 441, doi:10.1038/nature04794 Full text | PDF | Subscribe to Nature
The tumor suppressor gene PTEN is commonly mutated or deleted in human cancers, leading to the phosphorylation and activation of the proto-oncogene protein kinase AKT (PKB). Phosphorylated AKT inactivates several transcription factors in the nucleus, but little is known about its regulation in this compartment. In Nature, Pandolfi and colleagues now show that the tumor suppressor promyelocytic leukemia (Pml) keeps nuclear Akt in check.
, a member of the CNTF receptor complex, and AMPK activation are required for CNTF-dependent palmitate oxidation. Leptin and CNTF also activate signal transducer and activator of transcription (STAT3) to regulate the expression of gluconeogenic genes. It has previously been shown that leptin resistance is partly due to an upregulation of suppressor of cytokine signaling 3 (SOCS3) which inhibits the leptin-dependent activation of STAT3. However, in this study, Watt et al. show that STAT3 activation is not required for CNTF-induced AMPK signaling. 
In mitosis and meiosis, sister chromatids are bound together by the ring shaped cohesin complex. The centromeres of chromosomes assemble kinetochore complexes that mediate interaction with spindle microtubules. While cohesins detach from chromosome arms early in mitosis, cohesin around the centromere is dissolved much later, in anaphase, through cohesin cleavage by the protease separase. The persistence of centromeric cohesin requires a family of kinetochore localized proteins called the shugoshins. Two studies by Riedel et al. and Kitajima et al. in Nature now show that shugoshin (Sgo) recruits a subtype of protein phosphatase 2A (PP2A) to the centrosome, protecting cohesin from degradation. A further study by Brar et al. investigates whether phosphorylation of the meiosis specific cohesin subunit regulates the loss of centromeric cohesins.