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1.  Molecular Cloning and Characterization of Phocein, a Protein Found from the Golgi Complex to Dendritic Spines 
Molecular Biology of the Cell  2001;12(3):663-673.
Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the ς subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.
PMCID: PMC30971  PMID: 11251078
2.  The phocein homologue SmMOB3 is essential for vegetative cell fusion and sexual development in the filamentous ascomycete Sordaria macrospora 
Current Genetics  2011;57(2):133-149.
Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development.
Electronic supplementary material
The online version of this article (doi:10.1007/s00294-010-0333-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00294-010-0333-z
PMCID: PMC3059760  PMID: 21229248
Phocein; Hyphal fusion; Fruiting body development; Sordaria macrospora
3.  A Mammalian Homolog of Yeast MOB1 Is Both a Member and a Putative Substrate of Striatin Family-Protein Phosphatase 2A Complexes* 
The Journal of biological chemistry  2001;276(26):24253-24260.
Striatin and S/G2 nuclear autoantigen (SG2NA) are related proteins that contain membrane binding domains and associate with protein phosphatase 2A (PP2A) and many additional proteins that may be PP2A regulatory targets. Here we identify a major member of these complexes as class II mMOB1, a mammalian homolog of the yeast protein MOB1, and show that its phosphorylation appears to be regulated by PP2A. Yeast MOB1 is critical for cytoskeletal reorganization during cytokinesis and exit from mitosis. We show that mMOB1 associated with PP2A is not detectably phosphorylated in asynchronous murine fibroblasts. However, treatment with the PP2A inhibitor okadaic acid induces phosphorylation of PP2A-associated mMOB1 on serine. Moreover, specific inhibition of PP2A also results in hyperphosphorylation of striatin, SG2NA, and three unidentified proteins, suggesting that these proteins may also be regulated by PP2A. Indirect immunofluorescence produced highly similar staining patterns for striatin, SG2NA, and mMOB1, with the highest concentrations for each protein adjacent to the nuclear membrane. We also present evidence that these complexes may interact with each other. These data are consistent with a model in which PP2A may regulate mMOB1, striatin, and SG2NA to modulate changes in the cytoskeleton or interactions between the cytoskeleton and membrane structures.
doi:10.1074/jbc.M102398200
PMCID: PMC3503316  PMID: 11319234
4.  WD40 Repeat Proteins Striatin and S/G2 Nuclear Autoantigen Are Members of a Novel Family of Calmodulin-binding Proteins That Associate with Protein Phosphatase 2A* 
The Journal of biological chemistry  2000;275(8):5257-5263.
Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca2+-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G2 nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the postsynaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G2 phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca2+-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca2+-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.
PMCID: PMC3505218  PMID: 10681496
5.  PDCD10 Interacts with Ste20-related Kinase MST4 to Promote Cell Growth and Transformation via Modulation of the ERK Pathway 
Molecular Biology of the Cell  2007;18(6):1965-1978.
PDCD10 (programmed cell death 10, TFAR15), a novel protein associated with cell apoptosis has been recently implicated in mutations associated with Cerebral Cavernous Malformations (CCM). Yeast two-hybrid screening revealed that PDCD10 interacts with MST4, a member of Ste20-related kinases. This interaction was confirmed by coimmunoprecipitation and colocalization assays in mammalian cells. Furthermore, the co-overexpression of PDCD10 and MST4 promoted cell proliferation and transformation via modulation of the extracellular signal-regulated kinase (ERK) pathway. Potent short interfering RNAs (siRNAs) against PDCD10 (siPDCD10) and MST4 (siMST4) were designed to specifically inhibit the expression of PDCD10 and MST4 mRNA, respectively. The induction of siPDCD10 or siMST4 resulted in decreased expression of endogenous PDCD10 or MST4, which was accompanied by reduced ERK activity and attenuated cell growth and anchorage-independent growth. On the other hand, siMST4 had similar effects in PDCD10-overexpressed cells. And more importantly, we confirmed that either overexpressing or endogenous PDCD10 can increase the MST4 kinase activity in vitro. Our results demonstrated that PDCD10 modulation of ERK signaling was mediated by MST4, and PDCD10 could be a regulatory adaptor necessary for MST4 function, suggesting a link between cerebral cavernous malformation pathogenesis and the ERK-MAPK cascade via PDCD10/MST4.
doi:10.1091/mbc.E06-07-0608
PMCID: PMC1877091  PMID: 17360971
6.  Structural Comparison of Human Mammalian Ste20-Like Kinases 
PLoS ONE  2010;5(8):e11905.
Background
The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains.
Principal Findings
We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family.
Significance
The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design.
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doi:10.1371/journal.pone.0011905
PMCID: PMC2920948  PMID: 20730082
7.  Activation of the Mineralocorticoid Receptor Increases Striatin Levels 
American journal of hypertension  2011;25(2):243-249.
BACKGROUND
Aldosterone (ALDO), a critical regulator of sodium homeostasis, mediates its effects via activation of the mineralocorticoid receptor (MR) through mechanisms that are not entirely clear. Striatin, a membrane associated protein, interacts with estrogen receptors in endothelial cells.
METHODS
We studied the effects of MR activation in vitro and in vivo on striatin levels in vascular tissue.
RESULTS
We observed that dietary sodium restriction was associated with increased striatin levels in mouse heart and aorta and that striatin and MR are present in the human endothelial cell line, (EA.hy926), and in mouse aortic endothelial cells (MAEC). Further, we show that MR co-precipitates with striatin in vascular tissue. Incubation of EA.hy926 cells with ALDO (10−8 mol/l for 5–24 h) increases striatin protein and mRNA expression, an effect that was inhibited by canrenoic acid, an MR antagonist. Consistent with these observations, incubation of MAEC with ALDO increased striatin levels that were likewise blocked by canrenoic acid. To test the in vivo relevance of these findings, we studied two previously described mouse models of increased ALDO levels. Intraperitoneal ALDO administration augmented the abundance of striatin protein in mouse heart. We also observed that in a murine model of chronic ALDO-mediated cardiovascular damage following treatment with NG-nitro-L-arginine methyl ester plus angiotensin II an increased abundance of striatin protein in heart and kidney tissue.
CONCLUSION
Our results provide evidence that increased striatin levels is a component of MR activation in the vasculature and suggest that regulation of striatin by ALDO may modulate estrogen’s nongenomic effects.
doi:10.1038/ajh.2011.197
PMCID: PMC3773217  PMID: 22089104
aldosterone; angiotensin; animal physiology; antagonists; blood pressure; endothelial cells; heart tissue; hypertension; inflammation; L-NAME; mineralocorticoid receptor; RAAS
8.  Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ 
PLoS ONE  2012;7(1):e30983.
The mammalian ste20 kinase (MST) signaling pathway plays an important role in the regulation of apoptosis and cell cycle control. We sought to understand the role of MST2 kinase and Salvador homolog 1 (SAV1), a scaffolding protein that functions in the MST pathway, in adipocyte differentiation. MST2 and MST1 stimulated the binding of SAV1 to peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that plays a key role in adipogenesis. The interaction of endogenous SAV1 and PPARγ was detected in differentiating 3T3-L1 adipocytes. This binding required the kinase activity of MST2 and was mediated by the WW domains of SAV1 and the PPYY motif of PPARγ. Overexpression of MST2 and SAV1 increased PPARγ levels by stabilizing the protein, and the knockdown of SAV1 resulted in a decrease of endogenous PPARγ protein in 3T3-L1 adipocytes. During the differentiation of 3T3-L1 cells into adipocytes, MST2 and SAV1 expression began to increase at 2 days when PPARγ expression also begins to increase. MST2 and SAV1 significantly increased PPARγ transactivation, and SAV1 was shown to be required for the activation of PPARγ by rosiglitazone. Finally, differentiation of 3T3-L1 cells was augmented by MST2 and SAV1 expression and inhibited by knockdown of MST1/2 or SAV1. These results suggest that PPARγ activation by the MST signaling pathway may be a novel regulatory mechanism of adipogenesis.
doi:10.1371/journal.pone.0030983
PMCID: PMC3266932  PMID: 22292086
9.  CTTNBP2, but not CTTNBP2NL, regulates dendritic spinogenesis and synaptic distribution of the striatin–PP2A complex 
Molecular Biology of the Cell  2012;23(22):4383-4392.
Although both CTTNBP2 and CTTNBP2NL interact with cortactin and striatin/zinedin, CTTNBP2, but not CTTNBP2NL, is predominantly expressed in neurons and regulates dendritic spine distribution of cortactin and striatin/zinedin. The finding may be relevant to the association of CTTNBP2 with autism.
Cortactin-binding protein 2 (CTTNBP2) interacts with cortactin to regulate cortactin mobility and control dendritic spine formation. CTTNBP2 has also been associated with autistic spectrum disorder. The regulation of dendritic spinogenesis could explain the association of CTTNBP2 with autism. Sequence comparison has indicated that CTTNBP2 N-terminal–like protein (CTTNBP2NL) is a CTTNBP2 homologue. To confirm the specific effect of CTTNBP2 on dendritic spinogenesis, here we investigate whether CTTNBP2NL has a similar function to CTTNBP2. Although both CTTNBP2 and CTTNBP2NL interact with cortactin, CTTNBP2NL is associated with stress fibers, whereas CTTNBP2 is distributed to the cortex and intracellular puncta. We also provide evidence that CTTNBP2, but not CTTNBP2NL, is predominantly expressed in the brain. CTTNBP2NL does not show any activity in the regulation of dendritic spinogenesis. In addition to spine morphology, CTTNBP2 is also found to regulate the synaptic distribution of striatin and zinedin (the regulatory B subunits of protein phosphatase 2A [PP2A]), which interact with CTTNBP2NL in HEK293 cells. The association between CTTNBP2 and striatin/zinedin suggests that CTTNBP2 targets the PP2A complex to dendritic spines. Thus we propose that the interactions of CTTNBP2 and cortactin and the PP2A complex regulate spine morphogenesis and synaptic signaling.
doi:10.1091/mbc.E12-05-0365
PMCID: PMC3496612  PMID: 23015759
10.  A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons 
The Journal of Cell Biology  1996;134(4):1051-1062.
A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD- repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)- signaling pathway specific to excitatory synapses.
PMCID: PMC2120968  PMID: 8769426
11.  Co-operation between Raf-1 and Akt pathways for the inhibition of RASSF1A-MST2-triggered apoptosis 
Cancer research  2010;70(3):1195-1203.
Mammalian sterile20-like kinases (MST1/2) are involved in stress-induced apoptosis signalling. MST2 is inhibited by Raf-1 binding, and its activation requires dissociation from Raf-1 and binding to the RASSF1A tumour suppressor protein. Here, we have investigated the regulation of MST2 by the pro-survival phosphoinositide-3 kinase (PI3K)-Akt pathway. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras expression or depletion of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We identified two Akt phosphorylation sites (T117 and T384) in MST2. Mutation of these sites individually reduced phosphorylation, while the double mutation abolished it. These mutations, especially the double mutation, inhibited MST2 interactions with Raf-1, but enhanced binding to RASSF1A resulting in higher activation of downstream stress signalling pathways (JNK and p38 MAPK) and apoptosis. Biochemical and in situ FLIM experiments revealed a dual mechanism of inhibition. Akt phosphorylation of MST2 (i) blocks binding to RASSF1A and promotes sequestration into the inhibitory complex with Raf-1; and (ii) prevents MST2 homo-dimerisation which is essential for MST2 activation. Our results further show that the dissociation of the Raf-1-MST2 complex is part of mitogenic signalling, thereby linking induction of proliferation with the risk of apoptosis. Results with Ras effector domain mutants that selectively couple to either PI3K or Raf-1 show that Akt activation is necessary to abrogate MST2 activation in response to mitogenic stimulation. Thus, MST2 serves as a hub to integrate the biological outputs of the Raf-1 and Akt pathways.
doi:10.1158/0008-5472.CAN-09-3147
PMCID: PMC2880716  PMID: 20086174
MST2; Raf-1; Akt; crosstalk; apoptosis
12.  Okadaic Acid: A Tool to Study the Hippo Pathway 
Marine Drugs  2013;11(3):896-902.
Mammalian Ste20-like kinases 1 and 2 (MST1 and MST2) are activated in NIH3T3 cells exposed to okadaic acid. The Hippo pathway is a newly emerging signaling that functions as a tumor suppressor. MST1 and MST2 work as core kinases of the Hippo pathway and their activities depend on the autophosphorylation, which is negatively regulated by protein phosphatase 2A (PP2A). Okadaic acid has been frequently used to enhance the phosphorylation of MST1 and MST2 and to trigger the activation of the Hippo pathway. However other components of the Hippo pathway could also be targets of okadaic acid. In this review we first briefly summarize the molecular architecture of the Hippo pathway for the reference of researchers outside the field. We explain how MST kinases are regulated by PP2A and how okadaic acid activates MST2. Thereafter we discuss which components of the Hippo pathway are candidate substrates of protein phosphatases and which points we need to consider in the usage of okadaic acid to study the Hippo pathway.
doi:10.3390/md11030896
PMCID: PMC3705378  PMID: 23493077
Hippo pathway; kinase; okadaic acid; phosphatase
13.  Regulation of NDR Protein Kinase by Hydrophobic Motif Phosphorylation Mediated by the Mammalian Ste20-Like Kinase MST3†  
Molecular and Cellular Biology  2005;25(24):11019-11029.
NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.
doi:10.1128/MCB.25.24.11019-11029.2005
PMCID: PMC1316964  PMID: 16314523
14.  Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen 
Molecular Biology of the Cell  2001;12(1):185-199.
Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.
PMCID: PMC30577  PMID: 11160832
15.  Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with Proapoptotic Kinase Mst1 to Promote Cardiomyocyte Apoptosis 
PLoS ONE  2013;8(3):e58697.
Mammalian sterile 20-like kinase 1 (Mst1) is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.
doi:10.1371/journal.pone.0058697
PMCID: PMC3604124  PMID: 23527007
16.  Crucial Role for Mst1 and Mst2 Kinases in Early Embryonic Development of the Mouse▿  
Molecular and Cellular Biology  2009;29(23):6309-6320.
Mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2, respectively) are potent serine/threonine kinases that are involved in cell proliferation and cell death. To investigate the physiological functions of Mst1 and Mst2, we generated Mst1 and Mst2 mutant mice. Mst1−/− and Mst2−/− mice were viable and fertile and developed normally, suggesting possible functional overlaps between the two genes. A characterization of heterozygous and homozygous combinations of Mst1 and Mst2 mutant mice showed that mice containing a single copy of either gene underwent normal organ development; however, Mst1−/−; Mst2−/− mice lacking both Mst1 and Mst2 genes started dying in utero at approximately embryonic day 8.5. Mst1−/−; Mst2−/− mice exhibited severe growth retardation, failed placental development, impaired yolk sac/embryo vascular patterning and primitive hematopoiesis, increased apoptosis in placentas and embryos, and disorganized proliferating cells in the embryo proper. These findings indicate that both Mst1 and Mst2 kinases play essential roles in early mouse development, regulating placental development, vascular patterning, primitive hematopoiesis, and cell proliferation and survival.
doi:10.1128/MCB.00551-09
PMCID: PMC2786698  PMID: 19786569
17.  A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells 
Human Molecular Genetics  2008;18(5):911-918.
Cavernous vascular malformations occur with a frequency of 1:200 and can cause recurrent headaches, seizures and hemorrhagic stroke if located in the brain. Familial cerebral cavernous malformations (CCMs) have been associated with germline mutations in CCM1/KRIT1, CCM2 or CCM3/PDCD10. For each of the three CCM genes, we here show complete localized loss of either CCM1, CCM2 or CCM3 protein expression depending on the inherited mutation. Cavernous but not adjacent normal or reactive endothelial cells of known germline mutation carriers displayed immunohistochemical negativity only for the corresponding CCM protein but not for the two others. In addition to proving loss of function at the protein level, our data are the first to demonstrate endothelial cell mosaicism within cavernous tissues and provide clear pathogenetic evidence that the endothelial cell is the cell of disease origin.
doi:10.1093/hmg/ddn420
PMCID: PMC2640205  PMID: 19088124
18.  Novel KRIT1/CCM1 mutation in a patient with retinal cavernous hemangioma and cerebral cavernous malformation 
Backround
Retinal cavernous hemangiomas are rare vascular anomalies, and can be associated with cerebral cavernous malformations (CCM). Distinct mutations have been reported in patients who have both CCMs and retinal cavernous hemangiomas.
Methods
Fluorescein angiography, spectral domain optical coherence tomography, and genetic testing were performed on a patient with a retinal cavernous hemangioma and a CCM.
Results
Our patient was heterozygous in the KRIT1/CCM1 gene for a frameshift mutation, c.1088delC. This would be predicted to result in premature protein termination.
Discussion
We have identified a novel mutation in the KRIT1/CCM1 gene in a patient with both CCM and retinal cavernous hemangioma. We hypothesize that the occurrence of retinal cavernous hemangiomas and CCMs is underlaid by a common mechanism present in the KRIT1/CCM1 gene.
doi:10.1007/s00417-010-1329-6
PMCID: PMC2910301  PMID: 20306072
Cerebral cavernous malformation; KRIT1; Mutation; Retinal cavernous hemangioma
19.  The human WW45 protein enhances MST1-mediated apoptosis in vivo 
Mammalian sterile 20–like kinase 1 (MST1) is a serine/threonine protein kinase that is activated in response to a wide variety of apoptotic stimuli and can cause apoptosis when over-expressed in mammalian cells. The physiological regulation and cellular targets of MST1 are not well understood. Using a yeast two-hybrid system, we identified human WW45 (hWW45, also called hSav1) as an MST1-binding protein. The association between the two proteins was confirmed by immunofluorescence and co-immunoprecipitation, and hWW45 was present in both the cytoplasm and nucleus. When hWW45 alone was over-expressed, it weakly induced apoptosis. However, hWW45 augmented MST1-induced apoptosis when the two were co-expressed. Conversely, RNA interference-mediated depletion of endogenous hWW45 suppressed MST1-induced apoptosis. These results indicate that hWW45 is required to enhance MST1-mediated apoptosis in vivo and thus is a critical player in an MST1-driven cell death signaling pathway.
PMCID: PMC2851627  PMID: 19212654
hWW45; MST1; apoptosis
20.  CCM3 signaling through sterile 20–like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations 
The Journal of Clinical Investigation  2010;120(8):2795-2804.
Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Δ) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Δ proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20–like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.
doi:10.1172/JCI39679
PMCID: PMC2912181  PMID: 20592472
21.  A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3) 
Biochemical Journal  2012;442(Pt 3):595-610.
The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr178) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr328) in the regulatory domain, an event also requiring the MST3(341–376) sequence which acts as a putative docking domain. MST3(Thr178) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr328) phosphorylation. Interestingly, MST3(Thr328) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr178/Thr328) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr328) phosphorylation was necessary for formation of the activated MST3–MO25 holocomplex.
doi:10.1042/BJ20112000
PMCID: PMC3286863  PMID: 22229648
cardiac myocyte; germinal centre kinase; mammalian Sterile20-related kinase 3 (MST3); MO25; phosphorylation; regulation; AdV, adenovirus; CCM3, cerebral cavernous malformation 3; ECL, enhanced chemiluminescence; GCK, germinal centre kinase; GOLGA2, golgin A2; gm130, Golgi matrix protein 130; GST, glutathione transferase; HA, haemagglutinin; LKB1, liver kinase B1; mAb, monoclonal antibody; MASK, MST3 and SOK1-related kinase; MBP, myelin basic protein; MST, mammalian Sterile20-related kinase; NLS, nuclear localization signal; OXSR1, oxidative-stress responsive 1; PAK, p21-activated kinase; PASK, proline-alanine-rich Sterile20-related kinase; PDCD10, programmed cell death 10; PKA, cAMP-dependent protein kinase; PP, protein serine/threonine phosphatase; SOK1, Sterile20/oxidant stress response kinase1; SPAK, Sterile20/Sps1-related proline/alanine-rich kinase; Ste20, Sterile20; STRAD, Ste20-related adapter; TBST, Tris-buffered saline plus Tween 20; YSK1, yeast Sps1/Ste20-related kinase 1
22.  Rapid Estrogen Receptor Signaling is Essential for the Protective Effects of Estrogen Against Vascular Injury 
Circulation  2012;126(16):1993-2004.
Background
Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which when bound to estrogen can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a “rapid” non-nuclear signaling cascade. However, the biologic significance of this rapid signaling pathway has been unclear.
Methods and Results
Here, we develop a novel transgenic mouse in which rapid signaling is blocked by over-expression of a peptide that prevents ERs from interacting with the scaffold protein, striatin (the Disrupting Peptide Mouse, DPM). Microarray analysis of ex vivo-treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the DPM mouse following carotid artery wire injury.
Conclusions
Taken together, these results support that rapid, non-nuclear ER signaling contributes to the transcriptional regulatory functions of ER, and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.
doi:10.1161/CIRCULATIONAHA.112.124529
PMCID: PMC3780602  PMID: 22997253
cardiovascular diseases; hormones; molecular biology; signal transduction
23.  RASSF2 associates with and stabilises the proapoptotic kinase MST2 
Oncogene  2009;28(33):2988-2998.
RASSF2 is a tumour suppressor that in common with the rest of the RASSF family contains Ras association and SARAH domains. We identified the proapoptotic kinases MST1 and MST2 as the most significant binding partners of RASSF2, confirmed the interactions at endogenous levels and demonstrated that RASSF2 immunoprecipitates active MST1/2. We then demonstrated that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase which is likely to be MST1/2. Furthermore, we demonstrated that RASSF2 and MST2 do indeed colocalise, but whilst RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalisation in the cytoplasm. Expression of RASSF2 (stably in MCF7 or transiently in HEK-293) increases MST2 levels and knockdown of RASSF2 in HEK-293 cells reduces MST2 levels, additionally colorectal tumour cell lines and primary tumours with low RASSF2 levels show decreased MST2 protein levels. This is likely to be mediated by RASSF2-dependent protection of MST2 against proteolytic degradation. Our findings suggest that MST2 and RASSF2 form an active complex in vivo where RASSF2 is maintained in a phosphorylated state and protects MST2 from degradation and turnover. Thus we propose that the frequent loss of RASSF2 in tumours results in destabilisation of MST2 and thus decreased apoptotic potential.
doi:10.1038/onc.2009.152
PMCID: PMC2829092  PMID: 19525978
RASSF2; MST2; MST1; proteomics; epigenetics
24.  Towards the Development of a Potent and Selective Organoruthenium Mammalian Sterile 20 Kinase Inhibitor 
Journal of medicinal chemistry  2009;52(6):1602-1611.
Mammalian sterile 20 (MST1) kinase, a member of the sterile 20 (Ste-20) family of proteins, is a proapoptotic cytosolic kinase that plays an important role in the cellular response to oxidative stress. In this study, we report on the development of a potent and selective MST1 kinase inhibitor based on a ruthenium half-sandwich scaffold. We show that the enantiopure organoruthenium inhibitor, 9E1, has an IC50 value of 45 nM for MST1 and a greater than 25-fold inhibitor selectivity over the related Ste-20 kinases, p21 activated kinase 1 (PAK1), and p21 activated kinase 4 (PAK4) and an almost 10-fold selectivity over the related Thousand and one amino acids kinase 2 (TAO2). Compound 9E1 also displays a promising selectivity profile against unrelated protein kinases, however, the proto-oncogene serine/threonine protein kinase PIM1 (PIM-1) and glycogen synthase kinase 3 (GSK-3β) are inhibited with IC50 values in the low nanomolar range. We also show that 9E1 can inhibit MST1 function in cells. A cocrystal structure of a related compound with PIM-1 and a homology model with MST1 reveals the binding mode of this scaffold to MST1 and provides a starting point for the development of improved MST1 kinase inhibitors for possible therapeutic application.
doi:10.1021/jm8005806
PMCID: PMC2708071  PMID: 19226137
25.  Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus 
FEBS letters  2012;587(3):272-277.
Cerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM. We identify a previously uncharacterized domain at the C-terminus of CCM2 and determine its 1.9 Å resolution crystal structure. Because this domain is structurally homologous to the N-terminal domain of harmonin, we name it the CCM2 harmonin-homology domain or HHD. CCM2 HHD is observed in two conformations, and we employ analytical ultracentrifugation to test its oligomerization. Additionally, CCM2 HHD contains an unusually long 13-residue 310 helix. This study provides the first structural characterization of CCM2.
doi:10.1016/j.febslet.2012.12.011
PMCID: PMC3558538  PMID: 23266514
Protein-protein interaction; Signal transduction; Cerebral cavernous malformation; X-ray crystallography; harmonin-homology domain

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