Stress-activated, pro-apoptotic kinase which, following caspase-cleavage, enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. Key component of the Hippo signaling pathway which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ. Phosphorylation of YAP1 by LATS2 inhibits its translocation into the nucleus to regulate cellular genes important for cell proliferation, cell death, and cell migration. STK3/MST2 and STK4/MST1 are required to repress proliferation of mature hepatocytes, to prevent activation of facultative adult liver stem cells (oval cells), and to inhibit tumor formation. Phosphorylates NKX2-1 (By similarity). Phosphorylates NEK2 and plays a role in centrosome disjunction by regulating the localization of NEK2 to centrosome, and its ability to phosphorylate CROCC and CEP250. In conjunction with SAV1, activates the transcriptional activity of ESR1 through the modulation of its phosphorylation. Positively regulates RAF1 activation via suppression of the inhibitory phosphorylation of RAF1 on 'Ser-259'. Phosphorylates MOBKL1A and RASSF2. Phosphorylates MOBKL1B on 'Thr-74'. Acts cooperatively with MOBKL1B to activate STK38.
During interphase, centrosomes are held together by a proteinaceous linker that connects the proximal ends of the mother and daughter centriole. This linker is disassembled at the onset of mitosis in a process known as centrosome disjunction, thereby facilitating centrosome separation and bipolar spindle formation. The NIMA (never in mitosis A)-related kinase Nek2A is implicated in disconnecting the centrosomes through disjoining the linker proteins C-Nap1 and rootletin. However, the mechanisms controlling centrosome disjunction remain poorly understood. Here, we report that two Hippo pathway components, the mammalian sterile 20-like kinase 2 (Mst2) and the scaffold protein Salvador (hSav1), directly interact with Nek2A and regulate its ability to localize to centrosomes, and phosphorylate C-Nap1 and rootletin. Furthermore, we demonstrate that the hSav1-Mst2-Nek2A centrosome disjunction pathway becomes essential for bipolar spindle formation on partial inhibition of the kinesin-5 Eg5. We propose that hSav1-Mst2-Nek2A and Eg5 have distinct, but complementary functions, in centrosome disjunction.
Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
Mammalian MST2 kinase plays an important role in cell proliferation, survival, and apoptosis. In search of interacting proteins of MST2, we found that estrogen receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2 and hSAV leads to ligand-independent activation of ERα in human breast cancer MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary and sufficient for the induction of ERα transactivation. The expression of hSAV and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40) expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.
MST1 and MST2 are the mammalian Ste20-related protein kinases most closely related to Drosophila Hippo, a major regulator of cell proliferation and survival during development. Overexpression of MST1 or MST2 in mammalian cells is proapototic; however, little is known concerning the physiologic regulation of the endogenous MST1/MST2 kinases, their role in mammalian cell proliferation, or the identity of the MST1/MST2 substrates critical to proliferative regulation.
Proc. Natl. Acad. Sci. U.S.A. 93, 10099-10104 (1996)[PubMed:8816758]
The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.
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 showed that RASSF2 immunoprecipitates active MST1/2. We then showed that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase that is likely to be MST1/2. Furthermore, we showed that RASSF2 and MST2 do indeed colocalize, but whereas RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalization 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, in addition 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, in which 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 the destablization of MST2 and thus decreased apoptotic potential.
Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.
Many tumor suppressor proteins act to blunt the effects of mitogenic signaling pathways. Loss of function mutations in the merlin tumor suppressor underlie neurofibromatosis type 2 (NF2), a familial autosomal dominant cancer syndrome. Studies of Drosophila suggest that Hippo (hpo) is required for inhibition of cell proliferation mediated by dMer, the orthologue of human merlin. Mammalian sterile 20-like kinase-2 (Mst2) is a mammalian Hpo orthologue, and numerous studies implicate Mst2 as a tumor suppressor. Mst2 is negatively regulated by the proto-oncoprotein Raf-1 in a manner independent of the kinase activity of Raf-1. We sought to determine whether, in mammalian cells, merlin could positively regulate Mst2. We also sought to determine whether Mst2, in addition to being negatively regulated by Raf-1, might itself reciprocally regulate Raf-1. In contrast to findings from Drosophila, we find no evidence that mammalian merlin positively regulates mammalian Mst2. Instead, surprisingly, RNA interference silencing of Mst2 leads to elevated inhibitory phosphorylation of Raf-1 at Ser-259 and impaired Raf-1 kinase activity. Consequent to this, ERK pathway activation and cell proliferation are attenuated. Phosphatase-2A (PP2A) dephosphorylates Raf-1 Ser-259 in response to mitogens. Interestingly RNA interference silencing of Mst2 triggers a striking proteasome-dependent decrease in the levels of the catalytic subunit of PP2A (PP2A-C). A similar effect is achieved upon silencing of large tumor suppressor (LATS)-1 and LATS2, direct substrates of Mst2. Our studies reveal a more complex role for Mst2 than previously thought. The Mst2 --> LATS1/2 pathway, by maintaining PP2A-C levels, may, in some situations, positively affect mitogenic signaling.
Originally identified in Drosophila melanogaster, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Here we report that human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis revealed that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction was observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
Mammalian MST kinases function in stress-induced apoptosis to limit tumor progression. However, there is limited understanding about MST2 control by key regulators of cell division and survival. Raf-1 binds and inhibits MST2 kinase, whereas dissociation from Raf-1 and binding to tumor suppressor protein RASSF1A activates MST2. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras, or depletion of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10. We identified T117 and T384 as Akt phosphorylation sites in MST2. Mutation of these sites inhibited MST2 binding to Raf-1 kinase but enhanced binding to tumor suppressor RASSF1A, accentuating downstream c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signaling and promoting apoptosis. We determined that MST2 phosphorylation by Akt limits MST2 activity in two ways: first, by blocking its binding to RASSF1A and by promoting its association into the Raf-1 inhibitory complex, and second, by preventing homodimerization of MST2, which is needed for its activation. Dissociation of the Raf-1-MST2 complex promoted mitogenic signaling and coordinately licensed apoptotic risk. Using Ras effector domain mutants, we found that Akt is essential to prevent MST2 activation after mitogenic stimulation. Our findings elucidate how MST2 serves as a hub to integrate biological outputs of the Raf-1 and Akt pathways.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Mammalian MST2 kinase plays an important role in cell proliferation, survival, and apoptosis. In search of interacting proteins of MST2, we found that estrogen receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2 and hSAV leads to ligand-independent activation of ERα in human breast cancer MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary and sufficient for the induction of ERα transactivation. The expression of hSAV and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40) expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.
Evidence
3:
Inferred from Physical InteractionIntAct
Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.
Evidence
4:
Inferred from Physical InteractionIntAct
Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.
Evidence
5:
Inferred from Physical InteractionIntAct
The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.
Evidence
6:
Inferred from Physical InteractionUniProtKB
Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.
Evidence
7:
Inferred from Physical InteractionUniProtKB
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 showed that RASSF2 immunoprecipitates active MST1/2. We then showed that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase that is likely to be MST1/2. Furthermore, we showed that RASSF2 and MST2 do indeed colocalize, but whereas RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalization 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, in addition 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, in which 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 the destablization of MST2 and thus decreased apoptotic potential.
Evidence
8:
Inferred from Physical InteractionIntAct
Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.
Evidence
9:
Inferred from Physical InteractionIntAct
Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro-apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C-terminal coiled-coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled-coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N-terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C-terminal coiled-coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.
Evidence
10:
Inferred from Physical InteractionIntAct
Epigenetic inactivation of the candidate tumor suppressor gene RASSF1A is a frequent and critical event in the pathogenesis of many human cancers. The RASSF1A protein contains a Ras association domain, suggesting a role in Ras-like signaling pathways, and has also been implicated in cell cycle progression. However, the preliminary data suggests that the RASSF1A gene product is likely to have multiple functions. To identify novel RASSF1A functions, we have sought to identify interacting proteins by yeast two-hybrid analysis in a human brain cDNA library. We identified the E1A-regulated transcription factor p120(E4F) as a RASSF1A interacting partner in yeast and mammalian cells, and demonstrated that RASSF1A protein and p120(E4F) form a complex in vivo. The interaction between RASSF1A and p120(E4F) was confirmed by both in vitro and in vivo pull downs and coimmunoprecipitation assays. In addition, specific inactivation of RASSF1A by short interfering RNA disrupts binding of RASSF1A to p120(E4F) in coimmunoprecipitation assays. In addition, we demonstrated enhanced G(1) cell cycle arrest and S phase inhibition by propidium iodide staining of p120(E4F) in the presence of RASSF1A. As p120(E4F) has been reported previously to interact with p14ARF, retinoblastoma, and p53, these findings provide an important link between the function of RASSF1A and other major human tumor suppressor genes.
Evidence
11:
Inferred from Physical InteractionUniProtKB
The RASSF1A tumor suppressor protein interacts with the pro-apoptotic mammalian STE20-like kinases MST1 and MST2 and induces their autophosphorylation and activation, but the mechanism of how RASSF1A activates MST1/2 is unclear. Okadaic acid treatment and PP2A knockdown promoted MST1/2 phosphorylation. Data from dephosphorylation assays and reduced activation of MST1/2 seen after RASSF1A depletion suggest that dephosphorylation of MST1/2 on Thr-183 and Thr-180 by PP2A is prevented by RASSF1A, shifting the balance of MST1/2 to the activated autophosphorylated form. In addition to preventing dephosphorylation, RASSF1A also stabilized the MST2 protein. Through binding to MST1/2, RASSF1A supports maintenance of MST1/2 phosphorylation, promoting an active state of the MST kinases and favoring induction of apoptosis. This is one of the first examples of a tumor suppressor acting as an inhibitor of a specific dephosphorylation pathway.
Evidence
12:
Inferred from Physical InteractionUniProtKB
Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.
We have identified a cell quiescence-specific 33-kDa cytoplasmic protein kinase (p33(QIK), Quiescence-Induced Kinase) based on induction of p33(QIK)-specific kinase activity of cells growth-arrested in the quiescent phase and deactivation upon entry into the cell cycle. Blockage of macromolecular synthesis prevents p33(QIK) from deactivation, indicating a requirement of newly synthesized regulators for deactivation of p33(QIK) during G(0)/G(1) transition. Stress shock induces additional increases of p33(QIK) activity in a quiescence-dependent manner that correlates with induction of apoptosis. Using a specific antibody to Krs1/Mst2 protein, we found that p33(QIK) is related to p63(Krs1) and is distinguishable from a 36-kDa protein kinase, which is induced through proteolytic modification of activated p63(Krs1) in proliferating cells undergoing apoptosis. p33(QIK) is constantly expressed in quiescent, proliferating, and apoptotic quiescent cells. Regulation of p33(QIK) activity involves protein phosphorylation/dephosphorylation in a proteolysis-independent manner. Regulation of p33(QIK) and related p63(Krs1) and p36 appears to involve distinct pathways in quiescent and proliferating cells, respectively. Our results illustrate the relevance of p33(QIK) activity for cell quiescence that may provide a new insight into signaling pathways regulated in cells during quiescence and quiescence-related apoptosis.
Proc. Natl. Acad. Sci. U.S.A. 93, 10099-10104 (1996)[PubMed:8816758]
The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.
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 showed that RASSF2 immunoprecipitates active MST1/2. We then showed that RASSF2 can be phosphorylated by a co-immunoprecipitating kinase that is likely to be MST1/2. Furthermore, we showed that RASSF2 and MST2 do indeed colocalize, but whereas RASSF2 alone is nuclear, the presence of MST1 or MST2 results in colocalization 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, in addition 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, in which 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 the destablization of MST2 and thus decreased apoptotic potential.
MST1 and MST2 are the mammalian Ste20-related protein kinases most closely related to Drosophila Hippo, a major regulator of cell proliferation and survival during development. Overexpression of MST1 or MST2 in mammalian cells is proapototic; however, little is known concerning the physiologic regulation of the endogenous MST1/MST2 kinases, their role in mammalian cell proliferation, or the identity of the MST1/MST2 substrates critical to proliferative regulation.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
The human serine/threonine kinase, mammalian STE20-like kinase (MST), is considerably homologous to the budding yeast kinases, SPS1 and STE20, throughout their kinase domains. The cellular function and physiological activation mechanism of MST is unknown except for the proteolytic cleavage-induced activation in apoptosis. In this study, we show that MST1 and MST2 are direct substrates of caspase-3 both in vivo and in vitro. cDNA cloning of MST homologues in mouse and nematode shows that caspase-cleaved sequences are evolutionarily conserved. Human MST1 has two caspase-cleavable sites, which generate biochemically distinct catalytic fragments. Staurosporine activates MST either caspase-dependently or independently, whereas Fas ligation activates it only caspase-dependently. Immunohistochemical analysis reveals that MST is localized in the cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into the nucleus before nuclear fragmentation is initiated, suggesting it functions in the nucleus. Transiently expressed MST1 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm, which is independent of caspase activation. Furthermore, when stably expressed in HeLa cells, MST highly sensitizes the cells to death receptor-mediated apoptosis by accelerating caspase-3 activation. These findings suggest that MST1 and MST2 play a role in apoptosis both upstream and downstream of caspase activation.
The process whose specific outcome is the progression of the central nervous system over time, from its formation to the mature structure. The central nervous system is the core nervous system that serves an integrating and coordinating function. In vertebrates it consists of the brain, spinal cord and spinal nerves. In those invertebrates with a central nervous system it typically consists of a brain, cerebral ganglia and a nerve cord.
The process whose specific outcome is the progression of the endocardium over time, from its formation to the mature structure. The endocardium is an anatomical structure comprised of an endothelium and an extracellular matrix that forms the innermost layer of tissue of the heart, and lines the heart chambers.
The series of molecular signals mediated by the serine/threonine kinase Hippo or one of its orthologs. In Drosophila, Hippo in complex with the scaffold protein Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) transcriptional activator. The core fly components hippo, sav, wts and mats are conserved in mammals as STK4/3 (MST1/2), SAV1/WW45, LATS1/2 and MOB1.
The NDR/LATS family of kinases is a subgroup of the AGC group of protein kinases and is conserved from lower eukaryotes to humans. Like other AGC kinases, NDR/LATS kinases require phosphorylation of conserved Ser/Thr residues for activation. On the one hand, binding of the coactivator MOB to NDR/LATS allows autophosphorylation. On the other hand, MST kinases directly phosphorylate NDR/LATS kinases. In addition to our understanding of the molecular activation mechanisms, recent studies have shown that LATS kinases play a central role in Hippo/SWH (Salvador/Warts/Hippo) tumor suppressor pathways, which coordinate cell proliferation and apoptosis by regulating proto-oncogenes, such as YAP and TAZ. In this review, we summarize current knowledge of Merlin/MST/SAV/MOB/LATS/NDR/YAP/TAZ networks (also termed mammalian Hippo signaling) and their roles in mammalian cellular transformation.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
A series of reactions in which a signal is passed on to downstream proteins within the cell by sequential protein phosphorylation and activation of the cascade components.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
Any process that decreases the rate, frequency, or extent of the Wnt receptor signaling pathway through beta-catenin, the series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell, followed by propagation of the signal via beta-catenin, and ending with a change in transcription of target genes.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
Any process that activates or increases the frequency, rate or extent of the protein kinase B signaling cascade, a series of reactions mediated by the intracellular serine/threonine kinase protein kinase B.
Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.
Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.
Mammalian MST kinases function in stress-induced apoptosis to limit tumor progression. However, there is limited understanding about MST2 control by key regulators of cell division and survival. Raf-1 binds and inhibits MST2 kinase, whereas dissociation from Raf-1 and binding to tumor suppressor protein RASSF1A activates MST2. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras, or depletion of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10. We identified T117 and T384 as Akt phosphorylation sites in MST2. Mutation of these sites inhibited MST2 binding to Raf-1 kinase but enhanced binding to tumor suppressor RASSF1A, accentuating downstream c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signaling and promoting apoptosis. We determined that MST2 phosphorylation by Akt limits MST2 activity in two ways: first, by blocking its binding to RASSF1A and by promoting its association into the Raf-1 inhibitory complex, and second, by preventing homodimerization of MST2, which is needed for its activation. Dissociation of the Raf-1-MST2 complex promoted mitogenic signaling and coordinately licensed apoptotic risk. Using Ras effector domain mutants, we found that Akt is essential to prevent MST2 activation after mitogenic stimulation. Our findings elucidate how MST2 serves as a hub to integrate biological outputs of the Raf-1 and Akt pathways.
We have identified a second human homology of the yeast Ste20 protein kinase family, which we designate MST2. MST2 is most similar to the previously identified MST1 protein kinase (78% identity, 88% similarity). Northern analysis indicates that MST2 mRNA is expressed at high levels in adult kidney, skeletal and placental tissues and at very low levels in adult heart, lung, liver and brain tissues. An in vitro kinase assay indicates that MST2 can phosphorylate an exogenous substrate, as well as itself, and phospho-amino-acid analysis indicates that it is a serine/threonine protein kinase. The identification of MST2 suggests that there may be subfamilies of Ste20-like protein kinases and that MST1 and MST2 may define one of these subfamilies.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
Proc. Natl. Acad. Sci. U.S.A. 93, 10099-10104 (1996)[PubMed:8816758]
The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.
This protein acts as an enzyme. It is known to catalyze the following reaction
EC 2.7.11.1: ATP + a protein ⇄ ADP + a phosphoprotein.
CuratedUniProtKB
It requires the following cofactor
Magnesium.
CuratedUniProtKB
It is regulated in the following manner
Inhibited by the C-terminal non-catalytic region. Activated by caspase-cleavage. Full activation also requires homodimerization and autophosphorylation of Thr-180, which are inhibited by the proto-oncogene product RAF1. Activated by RASSF1 which acts by preventing its dephosphorylation.
The ablation of the protein kinase Raf-1 renders cells hypersensitive to apoptosis despite normal regulation of extracellular signal-regulated kinases, which suggests that apoptosis protection is mediated by a distinct pathway. We used proteomic analysis of Raf-1 signaling complexes to show that Raf-1 counteracts apoptosis by suppressing the activation of mammalian sterile 20-like kinase (MST2). Raf-1 prevents dimerization and phosphorylation of the activation loop of MST2 independently of its protein kinase activity. Depletion of MST2 from Raf-1-/- mouse or human cells abrogated sensitivity to apoptosis, whereas overexpression of MST2 induced apoptosis. Conversely, depletion of Raf-1 from Raf-1+/+ mouse or human cells led to MST2 activation and apoptosis. The concomitant depletion of both Raf-1 and MST2 prevented apoptosis.
The RASSF1A tumor suppressor protein interacts with the pro-apoptotic mammalian STE20-like kinases MST1 and MST2 and induces their autophosphorylation and activation, but the mechanism of how RASSF1A activates MST1/2 is unclear. Okadaic acid treatment and PP2A knockdown promoted MST1/2 phosphorylation. Data from dephosphorylation assays and reduced activation of MST1/2 seen after RASSF1A depletion suggest that dephosphorylation of MST1/2 on Thr-183 and Thr-180 by PP2A is prevented by RASSF1A, shifting the balance of MST1/2 to the activated autophosphorylated form. In addition to preventing dephosphorylation, RASSF1A also stabilized the MST2 protein. Through binding to MST1/2, RASSF1A supports maintenance of MST1/2 phosphorylation, promoting an active state of the MST kinases and favoring induction of apoptosis. This is one of the first examples of a tumor suppressor acting as an inhibitor of a specific dephosphorylation pathway.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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