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1.  The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation 
Journal of cell science  2008;121(0 23):3912-3921.
Summary
Nek6 and Nercc1/Nek9 belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase/metaphase arrest, and depletion of XNercc from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5, a kinesin necessary for spindle bipolarity. Nek6 phosphorylates Eg5 at several sites in vitro, and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. While Cdk1 phosphorylates nearly all Eg5 during mitosis at Thr926, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion arrests cells with a monopolar spindle; this can be rescued by Eg5 wildtype but not by Eg5(Thr926Ala). Eg5(Ser1033Ala) rescues half as well as wildtype whereas an Eg5(Ser1033Asp) mutant is nearly as effective. Thus Nek6 phosphorylates a subset of Eg5 polypeptides during mitosis at a conserved site, whose phosphorylation is critical for the mitotic function of Eg5.
doi:10.1242/jcs.035360
PMCID: PMC4066659  PMID: 19001501
2.  Hippo Signaling Regulates Differentiation and Maintenance in the Exocrine Pancreas 
Gastroenterology  2013;144(7):1543-1553.e1.
BACKGROUND & AIMS
The Hippo signaling pathway is a context-dependent regulator of cell proliferation, differentiation, and apoptosis in species ranging from Drosophila to humans. In this study, we investigated the role of the core Hippo kinases—Mst1 and Mst2—in pancreatic development and homeostasis.
METHODS
We used a Cre/LoxP system to create mice with pancreas-specific disruptions in Mst1 and Mst2 (Pdx1-Cre;Mst1−/−;Mst2fl/fl mice), the mammalian orthologs of Drosophila Hippo. We used a transgenic approach to overexpress Yap, the downstream mediator of Hippo signaling, in the developing pancreas of mice.
RESULTS
Contrary to expectations, the pancreatic mass of Pdx1-Cre;Mst1−/−;Mst2fl/fl mice was reduced compared with wild-type mice, largely because of postnatal de-differentiation of acinar cells into duct-like cells. Development of this phenotype coincided with postnatal reactivation of YAP expression. Ectopic expression of YAP during the secondary transition (a stage at which YAP is normally absent) blocked differentiation of the endocrine and exocrine compartments, whereas loss of a single Yap allele reduced acinar de-differentiation. The phenotype of Pdx1-Cre;Mst1−/−;Mst2fl/fl mice recapitulated cellular and molecular changes observed during chemical-induced pancreatitis in mice.
CONCLUSIONS
The mammalian Hippo kinases, and YAP, maintain postnatal pancreatic acinar differentiation in mice.
doi:10.1053/j.gastro.2013.02.037
PMCID: PMC3665616  PMID: 23454691
mouse model; signal transduction; Hpo; Wts
3.  The Ets transcription factor GABP is a novel component of the Hippo pathway essential for growth and antioxidant defense 
Cell reports  2013;3(5):10.1016/j.celrep.2013.04.020.
Summary
The transcriptional co-activator YAP plays an important role in organ size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced GSH depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects acetaminophen-induced liver injury. Similar to its effects on YAP, the Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.
doi:10.1016/j.celrep.2013.04.020
PMCID: PMC3855275  PMID: 23684612
GABP; Hippo pathway; YAP; Glutathione depletion; Liver injury; liver cancer
4.  Mammalian STE20-like kinase 2, not kinase 1, mediates photoreceptor cell death during retinal detachment 
Cell Death & Disease  2014;5(5):e1269-.
Photoreceptor cell death is the definitive cause of vision loss in retinal detachment (RD). Mammalian STE20-like kinase (MST) is a master regulator of both cell death and proliferation and a critical factor in development and tumorigenesis. However, to date the role of MST in neurodegeneration has not been fully explored. Utilizing MST1−/− and MST2−/− mice we identified MST2, but not MST1, as a regulator of photoreceptor cell death in a mouse model of RD. MST2−/− mice demonstrated significantly decreased photoreceptor cell death and outer nuclear layer (ONL) thinning after RD. Additionally, caspase-3 activation was attenuated in MST2−/− mice compared to control mice after RD. The transcription of p53 upregulated modulator of apoptosis (PUMA) and Fas was also reduced in MST2−/− mice post-RD. Retinas of MST2−/− mice displayed suppressed nuclear relocalization of phosphorylated YAP after RD. Consistent with the reduction of photoreceptor cell death, MST2−/− mice showed decreased levels of proinflammatory cytokines such as monocyte chemoattractant protein 1 and interleukin 6 as well as attenuated inflammatory CD11b cell infiltration during the early phase of RD. These results identify MST2, not MST1, as a critical regulator of caspase-mediated photoreceptor cell death in the detached retina and indicate its potential as a future neuroprotection target.
doi:10.1038/cddis.2014.218
PMCID: PMC4047884  PMID: 24874741
retinal detachment photoreceptor cell; apoptosis; neurodegeneration; mammalian STE20-like kinase
6.  Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging* 
The Journal of Biological Chemistry  2013;289(5):2658-2674.
Background: Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers.
Results: mTOR complex 1-Rag binding in vitro is independent of Rag guanyl nucleotide charging, and withdrawal of amino acids from cells does not alter Rag GTP charging.
Conclusion: Amino acids promote Rag binding to mTOR complex 1 without changing Rag GTP charging.
Significance: Amino acids promote Rag-mTORC1 binding by undefined mechanisms.
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to RasSer-17 abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from 32P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [32P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
doi:10.1074/jbc.M113.528505
PMCID: PMC3908400  PMID: 24337580
Cell Signaling; GTPase; Insulin; Lysosomes; mTOR Complex (mTORC); Rag GTPase; Amino Acids; Raptor
7.  Protein Kinases of the Hippo Pathway: Regulation and Substrates 
The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP, promotes integrin clustering, actin remodeling and motility while restraining the proliferation of naïve T cells. This review will summarize current knowledge of the structure and regulation of the kinases Hippo/Mst1&2, their noncatalytic binding partners, Salvador and the Rassf polypeptides, and their major substrates Warts/Lats1&2, Trc/ndr1&2, Mats/Mob1 and FOXO.
doi:10.1016/j.semcdb.2012.07.002
PMCID: PMC3489012  PMID: 22898666
Protein kinase; Hippo; Mst1/2; Mob1; Lats1/2; ndr1/2
8.  YAP oncogene overexpression supercharges colon cancer proliferation 
Cell Cycle  2012;11(6):1090-1096.
The transcriptional co-activator YAP is an evolutionarily conserved regulator of organ size and progenitor cell proliferation. YAP is overexpressed at high frequency in many common human cancers and can directly drive cancer development in mouse models. YAP abundance and nuclear localization are negatively regulated by the Hippo kinase cascade, which, in epithelia, is activated by physiological cell-cell contact. recent work in intestinal epithelium has established that YAP is constitutively inhibited by the Hippo pathway and entirely dispensable for normal development and homeostasis. YAP serves only in a standby capacity; should cell-cell contact be abrogated, as after intestinal damage, the loss of Hippo input permits increased YAP abundance and nuclear residence. in turn, YAP cooperates with β-catenin to transactivate genes that promote stem cell expansion for epithelial repair. This interplay between overexpressed YAP and β-catenin also drives proliferation of colon cancer cells. The dispensability of YAP in normal intestine makes YAP's expression or outputs attractive targets for cancer therapy.
doi:10.4161/cc.11.6.19453
PMCID: PMC3335916  PMID: 22356765
YAP; Hippo; intestinal stem cell; β-catenin; colon cancer; liver cancer
9.  A Genome-wide RNAi Screen for Polypeptides that Alter rpS6 Phosphorylation 
Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains 3–5% despite current nonspecific and targeted therapies. Although rapamycin-related mTOR inhibitors have yet to demonstrate encouraging clinical responses, it is now evident that this class of compounds is capable of only partial mTORC1 inhibition. Identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new targets and lead to the development of beneficial therapies for pancreatic cancer.
doi:10.1007/978-1-61779-430-8_11
PMCID: PMC3535007  PMID: 22125066
rpS6; Phosphorylation; mTOR; Genome wide; RNAi; Screen; Immunofluorescence; Pancreatic cancer
10.  The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes 
In mice lacking both Mst1 and Mst2 in the lymphoid compartment, thymocyte development is normal, but single-positive thymocytes exhibit excessive apoptosis and greatly diminished thymic egress, accompanied by loss of chemokine activation of RhoA and Rac1.
The Mst1 kinase is an important regulator of murine T cell adhesion, migration, proliferation, and apoptosis. In this study, we analyze mice lacking both Mst1 and Mst2 in hematopoietic cells. Compared with wild-type mice, these double knockout (DKO) mice exhibit a severe reduction in the number of mature T cells in the circulation and in secondary lymphoid organs (SLOs). CD4+CD8− and CD4−CD8+ single-positive (SP) thymocytes in DKO mice resemble mature T cells of wild-type mice but undergo excessive apoptosis, and their egress from the thymus is reduced by >90%. Even when placed directly in the circulation, DKO SP thymocytes failed to enter SLOs. In SP thymocytes, deficiency of Mst1 and Mst2 abolished sphingosine-1 phosphate– and CCL21-induced Mob1 phosphorylation, Rac1 and RhoA GTP charging, and subsequent cell migration. When phosphorylated by Mst1 or Mst2, Mob1 binds and activates the Rac1 guanyl nucleotide exchanger Dock8, which is abundant in the thymus. Thus, the Mst1 and Mst2 kinases control Rho GTPase activation and the migratory responses of SP thymocytes.
doi:10.1084/jem.20111692
PMCID: PMC3328371  PMID: 22412158
11.  Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes 
PLoS ONE  2012;7(8):e41756.
Background
No targeted immunotherapies reverse type 1 diabetes in humans. However, in a rodent model of type 1 diabetes, Bacillus Calmette-Guerin (BCG) reverses disease by restoring insulin secretion. Specifically, it stimulates innate immunity by inducing the host to produce tumor necrosis factor (TNF), which, in turn, kills disease-causing autoimmune cells and restores pancreatic beta-cell function through regeneration.
Methodology/Principal Findings
Translating these findings to humans, we administered BCG, a generic vaccine, in a proof-of-principle, double-blind, placebo-controlled trial of adults with long-term type 1 diabetes (mean: 15.3 years) at one clinical center in North America. Six subjects were randomly assigned to BCG or placebo and compared to self, healthy paired controls (n = 6) or reference subjects with (n = 57) or without (n = 16) type 1 diabetes, depending upon the outcome measure. We monitored weekly blood samples for 20 weeks for insulin-autoreactive T cells, regulatory T cells (Tregs), glutamic acid decarboxylase (GAD) and other autoantibodies, and C-peptide, a marker of insulin secretion. BCG-treated patients and one placebo-treated patient who, after enrollment, unexpectedly developed acute Epstein-Barr virus infection, a known TNF inducer, exclusively showed increases in dead insulin-autoreactive T cells and induction of Tregs. C-peptide levels (pmol/L) significantly rose transiently in two BCG-treated subjects (means: 3.49 pmol/L [95% CI 2.95–3.8], 2.57 [95% CI 1.65–3.49]) and the EBV-infected subject (3.16 [95% CI 2.54–3.69]) vs.1.65 [95% CI 1.55–3.2] in reference diabetic subjects. BCG-treated subjects each had more than 50% of their C-peptide values above the 95th percentile of the reference subjects. The EBV-infected subject had 18% of C-peptide values above this level.
Conclusions/Significance
We conclude that BCG treatment or EBV infection transiently modified the autoimmunity that underlies type 1 diabetes by stimulating the host innate immune response. This suggests that BCG or other stimulators of host innate immunity may have value in the treatment of long-term diabetes.
Trial Registration
ClinicalTrials.gov NCT00607230
doi:10.1371/journal.pone.0041756
PMCID: PMC3414482  PMID: 22905105
12.  Yap1 acts downstream of α-catenin to control epidermal proliferation 
Cell  2011;144(5):782-795.
During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that α-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. α-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a ‘crowd control’ molecular circuitry in mammalian skin.
doi:10.1016/j.cell.2011.02.031
PMCID: PMC3237196  PMID: 21376238
13.  Mst1/2 signalling to Yap: gatekeeper for liver size and tumour development 
British Journal of Cancer  2010;104(1):24-32.
The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans.
doi:10.1038/sj.bjc.6606011
PMCID: PMC3039822  PMID: 21102585
liver cancer; hepatocellular carcinoma; cholangiocarcinoma; oval cells; Hippo; Rassf polypeptides; tumour suppressor pathway
14.  The Rheb Switch 2 Segment Is Critical for Signaling to Target of Rapamycin Complex 1* 
The Journal of biological chemistry  2007;282(25):18542-18551.
The small GTPase Rheb is a positive upstream regulator of the target of rapamycin (TOR) complex 1 in mammalian cells and can bind directly to TOR complex 1. To identify the regions of the Rheb surface most critical for signaling to TOR complex 1, we created a set of 26 mutants wherein clusters of 1–5 putative solvent-exposed residues were changed to alanine, ultimately changing 65 residues distributed over the entire Rheb surface. The signaling function of these mutants was assessed by their ability, in comparison to wild type Rheb, to restore the phosphorylation of S6K1(Thr389) when expressed transiently in amino acid-deprived 293T cells. The major finding is that two mutants situated in the Rheb switch 2 segment, Y67A/I69A and I76A/D77A, exhibit a near total loss of function, whereas extensive replacement of the switch 1 segment and other surface residues with alanines causes relatively little disturbance of Rheb rescue of S6K1 from amino acid withdrawal. This is surprising in view of the minimal impact of guanyl nucleotide on Rheb switch 2 configuration. The loss of function Rheb switch 2 mutants are well expressed and exhibit partial agonist function in amino acid-replete cells. They are unimpaired in their ability to bind GTP or mammalian (m)TOR in vivo or in vitro, and the mTOR polypeptides retrieved with these inactive Rheb mutants exhibit kinase activity in vitro comparable with mTOR bound to wild type Rheb. We conclude that Rheb signaling to mTOR in vivo requires a Rheb switch 2-dependent interaction with an element other than the three known polypeptide components of TOR complex 1.
doi:10.1074/jbc.M610736200
PMCID: PMC3205911  PMID: 17470430
15.  Nore1B regulates TCR signaling via Ras and Carma1 
Cellular signalling  2006;18(10):1647-1654.
Nore1A was originally identified as a potential Ras effector, and Nore1B is an alternatively spliced isoform. Both share a Ras/Rap association domain (RA domain) but only Nore1A contains sequence motifs that predict SH3 domain binding and diacylglycerol/phorbol ester binding in the amino-terminal region. Here we report that Carma1 binds to Nore1A and Nore1B through the RA domain and that Carma1 interacts with active Ras in the presence of Nore1B. RNA interference against Nore1B attenuates NF-κB activation induced by T cell receptor (TCR) ligation, but not NF-κB activation induced by TNFα or lipoteichoic acid. In addition, Nore1B is also required for KiRas GV12-mediated ERK1 activation and Elk1 reporter activity in T cells. We also provide evidence that knockdown of Nore1B also impairs polarized redistribution of Ras at the B cell-T cell immune interface. Together, these findings suggest that endogenous Nore1B recruits active Ras to the APC-T cell interface and mediates the interaction between Ras and Carma1.
doi:10.1016/j.cellsig.2006.01.015
PMCID: PMC3204664  PMID: 16520020
T-lymphocytes; NF-κB; Ras proteins; MAGUK proteins; Lymphocyte activation
16.  pTOM, a vector for expressing two proteins in mammalian cells 
Analytical biochemistry  2009;388(2):353-355.
We developed a pTOM construct that can express two proteins in a cell. Using pTOM ensures that two polypeptide-encoding nucleotide sequences are simultaneously transfected into the same cell. The ability to simultaneously express two separate polypeptide-encoding nucleotide sequences from the same vector in the same cell allows the user to determine the relationship between the two proteins. Another advantage is that one of the proteins can be used as a transfection marker/selector. The vector contains multiple cloning sites for insertions of polypeptide-encoding nucleotide sequences. Positive clones can be easily selected when performing cloning using ampicillin. Overall, this vector provides a convenient way to express dual proteins in a single mammalian cell.
doi:10.1016/j.ab.2009.03.015
PMCID: PMC3197823  PMID: 19303393
17.  The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1*† 
The Journal of biological chemistry  2007;282(28):20329-20339.
The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser183). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe129 to Ala). Immediately carboxyl-terminal to Phe129 are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser183, to Asp. PRAS40 (Ser183) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser183) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overex-pressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser183) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.
doi:10.1074/jbc.M702636200
PMCID: PMC3199301  PMID: 17517883
18.  Mst1 and Mst2 maintain hepatocyte quiescence and suppress the development of hepatocellular carcinoma through inactivation of the Yap1 oncogene 
Cancer cell  2009;16(5):425-438.
Hippo-Lats-Yorkie signaling regulates tissue overgrowth and tumorigenesis in Drosophila. We show that the Mst1 and Mst2 protein kinases, the mammalian Hippo orthologs, are cleaved and constitutively activated in the mouse liver. Combined Mst1/2 deficiency in the liver results in loss of inhibitory Ser127 phosphorylation of the Yorkie ortholog, Yap1, massive overgrowth, and hepatocellular carcinoma (HCC). Reexpression of Mst1 in HCC-derived cell lines promotes Yap1 Ser127 phosphorylation and inactivation, and abrogates their tumorigenicity. Notably, Mst1/2 inactivates Yap1 in liver through an intermediary kinase distinct from Lats1/2. Approximately 30% of human HCCs show low Yap1(Ser127) phosphorylation and a majority exhibit loss of cleaved, activated Mst1. Mst1/2 inhibition of Yap1 is an important pathway for tumor suppression in liver relevant to human HCC.
Significance
The pathways that regulate quiescence and tumor suppression in the liver have not been fully elucidated. We show that the Mst1 and Mst2 kinases are tumor suppressors and regulators of liver size in adults and that negative regulation of the transcriptional coactivator, Yap1, is central to Mst1/2 tumor suppressor function. Loss of both Mst1 and Mst2 is sufficient to initiate hepatocyte proliferation, resulting in dramatic liver overgrowth, resistance to pro-apoptotic stimuli, and the development of HCC. Mst1 and Mst2 promote phosphorylation of Yap1 and thereby suppress its oncogenic activity. Mst1/2 regulation of Yap1 is tissue-specific and, in the liver, involves an Mst1/2-regulated Yap1 kinase distinct from Lats1/2. Significantly, the Mst-Yap1 pathway is disrupted in a substantial fraction of human HCCs.
doi:10.1016/j.ccr.2009.09.026
PMCID: PMC3023165  PMID: 19878874
Mst1; Mst2; hepatocellular carcinoma; tumor suppressor; Yap1; hippo
20.  The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner▿ ‡  
Molecular and Cellular Biology  2008;29(3):640-649.
The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.
doi:10.1128/MCB.00980-08
PMCID: PMC2630691  PMID: 19047368
21.  Characterization of Two Mst1-Deficient Mouse Models 
Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase belonging to the family of Sterile 20-like kinases. MST1 has been inferred to play important roles in apoptosis and in the inhibition of proliferation in mammalian cells. Here, we describe the genetic characterization of Mst1-deficient mice produced by two distinct gene-trap insertions. Animals generated from clone RRT293 exhibit transmission ratio distortion favoring the mutated allele and is amplified with each generation. Inexplicably, while the mutated allele is favored for transmission, its homozygosity is embryonic lethal. By contrast, animals generated from the second Mst1 gene-trap clone, AJ0315, do not show any gross abnormalities. We find that the discrepancy in phenotype is most likely attributable to a second insertion in the RRT293 clone. Thus, a mutation in Mst1 alone does not affect survival. Our results set the stage for identification of the lethal second-site mutation that is paradoxically favored for transmission.
doi:10.1002/dvdy.21764
PMCID: PMC2596583  PMID: 18942145
Mst1; Sterile 20-like kinases; transmission ratio distortion; gene-trap insertion
22.  A Rictor-Myo1c Complex Participates in Dynamic Cortical Actin Events in 3T3-L1 Adipocytes▿ ‡  
Molecular and Cellular Biology  2008;28(13):4215-4226.
Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.
doi:10.1128/MCB.00867-07
PMCID: PMC2447144  PMID: 18426911
23.  MAP kinase pathways: The first twenty years 
Biochimica et biophysica acta  2006;1773(8):1150-1160.
The MAP kinases, discovered approximately twenty years ago, together with their immediate upstream regulators, are among the most highly studied signal transduction molecules. This body of work has shaped many aspects of our present views of signal transduction by protein kinases. The effort expended in this area reflects the extensive participation of these regulatory modules in the control of cell fate decisions, i.e., proliferation, differentiation and death, across all eukaryotic phylla and in all tissues of metazoans. The discovery of these kinases is reviewed, followed by a discussion of some of the features of this signaling module that account for its broad impact on cell function and its enormous interest to many investigators.
doi:10.1016/j.bbamcr.2006.11.006
PMCID: PMC2043147  PMID: 17229475
24.  Getting a grip on waiting lists 
doi:10.1503/cmaj.1050180
PMCID: PMC1329467  PMID: 16415474
25.  Active Nercc1 Protein Kinase Concentrates at Centrosomes Early in Mitosis and Is Necessary for Proper Spindle Assembly 
Molecular Biology of the Cell  2005;16(10):4827-4840.
The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates γ-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains γ-tubulin and several components of the γ-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the γ-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.
doi:10.1091/mbc.E05-04-0315
PMCID: PMC1237086  PMID: 16079175

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