Mammalian Ste20-like protein kinase 3 mediates trophoblast apoptosis in spontaneous delivery

The placenta is essential in transferring gases and nutrients from the mother to the developing fetus. Trophoblast apoptosis may cause labor or other pregnancy-related disorders. This study demonstrated the essential role of Mst3, a human Ste20-like protein kinase, in the oxidative stress-induced apoptosis of trophoblasts of term placenta in normal spontaneous delivery. Oxidative stress, but not hormones released during labor such as prostaglandin E₁, oxytocin or angiotensin II, induces the expression of Mst3 and apoptosis of human term placenta after elective Cesarean section without labor pain. The role of Mst3 in oxidative stress-induced apoptosis was further demonstrated in the 3A-sub-E, a human trophoblast cell line. The H₂O₂-induced apoptosis of 3A-sub-E cells was largely suppressed by overexpressed Mst3^KR, the kinase-dead mutant or by selective knockdown of endogenous Mst3. Further studies showed that Jun N-terminal kinase (JNK) may participate in the signaling pathway of H₂O₂-induced apoptosis by mediating the level of Mst3. Subsequently, caspase 3 and other downstream apoptotic components may be activated by Mst3 and trigger the apoptotic process in human trophoblasts.     

Mammalian Ste20-like protein kinase 3 plays a role in hypoxia-induced apoptosis of trophoblast cell line 3A-sub-E

Mammalian Ste20-like protein kinase 3 (Mst3) is a key player in inducing apoptosis in a variety of cell types and has recently been shown to participate in the signaling pathway of hypoxia-induced apoptosis of human trophoblast cell line 3A-sub-E (3A). It is believed that oxidative stress may occur during hypoxia and induce the expression of Mst3 in 3A cells via the activation of c-Jun N-terminal protein kinase 1 (JNK1). This hypothesis was demonstrated by the suppressive effect of dl-α-lipoic acid, a reactive oxygen species scavenger, in hypoxia-induced responses of 3A cells such as Mst3 expression, nitrotyrosine formation, JNK1 activation and apoptosis. Similar results were also observed in trophoblasts of human placental explants in both immunohistochemical studies and immunoblot analyses. These suggested that the activation of Mst3 might trigger the apoptotic process in trophoblasts by activating caspase 3 and possibly other apoptotic pathways. The role of nitric oxide synthase (NOS) and NADPH oxidase (NOX) in hypoxia-induced Mst3 up-regulation was also demonstrated by the inhibitory effect of N(G)-nitro-l-arginine and apocynin, which inhibits NOS and NOX, respectively. Oxidative stress was postulated to be induced by NOS and NOX in 3A cells during hypoxia. In conclusion, hypoxia induces oxidative stress in human trophoblasts by activating NOS and NOX. Subsequently, Mst3 is up-regulated and plays an important role in hypoxia-induced apoptosis of human trophoblasts.     

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

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.     

Activation of a human Ste20-like kinase by oxidant stress defines a novel stress response pathway.

Mammalian homologs of the yeast protein kinase, Sterile 20 (Ste20), can be divided into two groups based on their regulation and structure. The first group, which includes PAK1, is regulated by Rac and Cdc42Hs, and activators have been identified. In contrast, very little is known about activators, regulatory mechanisms or physiological roles of the other group, which consists of GC kinase and MST1. We have identified a human Ste20-like kinase from the GC kinase group, SOK-1 (Ste20/oxidant stress response kinase-1), which is activated by oxidant stress. The kinase is activated by autophosphorylation and is markedly inhibited by its non-catalytic C-terminal region. SOK-1 is activated 3- to 7-fold by reactive oxygen intermediates, but is not activated by growth factors, alkylating agents, cytokines or environmental stresses including heat shock and osmolar stress. Although these data place SOK-1 on a stress response pathway, SOK-1, unlike GC kinase and PAK1, does not activate either of the stress-activated MAP kinase cascades (p38 and SAPKs). SOK-1 is the first mammalian Ste20-like kinase which is activated by cellular stress, and the activation is relatively specific for oxidant stress. Since SOK-1 does not activate any of the known MAP kinase cascades, its activation defines a novel stress response pathway which is likely to include a unique stress-activated MAP kinase cascade.     

Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

BACKGROUND The low survival rate of mesenchymal stem cells(MSCs) caused by anoikis, a form of apoptosis, limits the therapeutic efficacy of MSCs. As a proapoptotic molecule, mammalian Ste20-like kinase 1(Mst1) can increase the production of reactive oxygen species(ROS), thereby promoting anoikis. Recently, we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs) from H2O2-induced cell apoptosis by inducing autophagy and reducing ROS production. However, the influence of Mst1 in...     

v-Src-dependent down-regulation of the Ste20-like kinase SLK by casein kinase II

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein inducing actin stress fiber disassembly. Here, we show that v-Src expression can down-regulate SLK activity. This down-regulation is independent of focal adhesion kinase but requires v-Src kinase activity and membrane translocation. SLK down-regulation by v-Src is indirect and is accompanied by SLK hyperphosphorylation on serine residues. Deletion analysis revealed that casein kinase II (CK2) sites at position 347/348 are critical for v-Src-dependent modulation of SLK activity. Further studies show that CK2 can directly phosphorylate SLK at these positions and that inhibition of CK2 in v-Src-transformed cells results in normal kinase activity. Finally, CK2 and SLK can be co-localized in fibroblasts spreading on fibronectin-coated substrates, suggesting a mechanism whereby SLK may be regulated at sites of actin remodeling, such as membrane lamellipodia and ruffles, through CK2.     

Cloning and characterization of MST4, a novel Ste20-like kinase

MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase.     

Identification and characterization of the nuclear import and export signals of the mammalian Ste20-like protein kinase 3

Mst3, a human Ste20-like protein kinase, has been recently demonstrated to undergo a caspase-mediated cleavage during apoptosis. The proteolytic cleavage of the C-terminus of Mst3 caused nuclear translocation of its kinase domain. This work provides evidence that Mst3 may contain a bipartite-like nuclear localization sequence (NLS) at the C-terminus of its kinase domain (residues 278-292). The removal of NLS from the kinase domain of Mst3 led to the cytoplasmic accumulation of EGFP-Mst3(Delta277). The presence of nuclear exporting signals in the Mst3 was also demonstrated by leptomycin B-treatment and serial deletion of the C-terminal regulatory domain of Mst3. A nuclear export signal was also postulated to be in the regions of amino acids 335-386. In conclusion, Mst3 contains both NLS and NES signals, which may cooperate to control the subcellular distribution of Mst3.     

MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1

MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. We studied the activation of MARK and identified the upstream kinase, MARKK, a member of the Ste20 kinase family. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation.     

The Mammalian Ste20-like Kinase 2 (Mst2) Modulates Stress-induced Cardiac Hypertrophy

The Hippo signaling pathway has recently moved to center stage in cardiac research because of its key role in cardiomyocyte proliferation and regeneration of the embryonic and newborn heart. However, its role in the adult heart is incompletely understood. We investigate here the role of mammalian Ste20-like kinase 2 (Mst2), one of the central regulators of this pathway. Mst2^−/− mice showed no alteration in cardiomyocyte proliferation. However, Mst2^−/− mice exhibited a significant reduction of hypertrophy and fibrosis in response to pressure overload. Consistently, overexpression of MST2 in neonatal rat cardiomyocytes significantly enhanced phenylephrine-induced cellular hypertrophy. Mechanistically, Mst2 positively modulated the prohypertrophic Raf1-ERK1/2 pathway. However, activation of the downstream effectors of the Hippo pathway (Yes-associated protein) was not affected by Mst2 ablation. An initial genetic study in mitral valve prolapse patients revealed an association between a polymorphism in the human MST2 gene and adverse cardiac remodeling. These results reveal a novel role of Mst2 in stress-dependent cardiac hypertrophy and remodeling in the adult mouse and likely human heart.     

Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end–directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150^Glued subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150^Glued and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150^Glued to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150^Glued expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150^Glued suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.     

The SH2/SH3 adaptor protein dock interacts with the Ste20-like kinase misshapen in controlling growth cone motility

Recent studies suggest that the SH2/SH3 adaptor Dock/Nck transduces tyrosine phosphorylation signals to the actin cytoskeleton in regulating growth cone motility. The signaling cascade linking the action of Dock/Nck to the reorganization of cytoskeleton is poorly understood. We now demonstrate that Dock interacts with the Ste20-like kinase Misshapen (Msn) in the Drosophila photoreceptor (R cell) growth cones. Loss of msn causes a failure of growth cones to stop at the target, a phenotype similar to loss of dock, whereas overexpression of msn induces pretarget growth cone termination. Physical and genetic interactions between Msn and Dock indicate a role for Msn in the Dock signaling pathway. We propose that Msn functions as a key controller of growth cone cytoskeleton in response to Dock-mediated signals.     

Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.     

The Ste20-like kinase SLK is required for cell cycle progression through G2

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.     

Bioinformatics search for plant homologues of Ste20-like serine/threonine protein kinases

Eleven plant homologuess of animal and yeast STE20-like protein kinases were identified. It was shown that unknown proteins A9RVK0 of the moss Physcomitrella patens ssp. patens and A7P2E2 of the grape Vitis vinifera were the closest plant homologues of STE20-like protein kinases. The Ste20-like protein kinase dst1 of the myxomycete Dictyostelium discoideum was the closest to the plant homologues. The spatial structure of the catalytic domain of the protein A9RVK0 from P. patens ssp. patens was predicted.     

Induction of apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates apoptosis signal-regulating kinase and p38

Expression and activity of the germinal center kinase, Ste20-like kinase (SLK), are increased during kidney development and recovery from ischemic acute renal failure. In this study, we characterize the activation and functional role of SLK. SLK underwent dimerization via the C-terminal domain, and dimerization enhanced SLK activity. In contrast, the C-terminal domain of SLK did not dimerize with a related kinase, Mst1, and did not affect Mst1 activity. Phosphorylation/dephosphorylation of SLK were not associated with changes in kinase activity. SLK induced phosphorylation of apoptosis signal-regulating kinase-1 (ASK1) and increased ASK1 activity, indicating that ASK1 is a substrate of SLK. Moreover, SLK stimulated phosphorylation of p38 mitogen-activated protein kinase via ASK1, but not c-Jun N-terminal kinase nor extracellular signal-regulated kinase. Chemical anoxia and recovery during re-exposure to glucose (ischemia-reperfusion injury in cell culture) stimulated SLK activity. Overexpression of SLK enhanced anoxia/recovery-induced apoptosis, release of cytochrome c, and activities of caspase-8 and -9, and apoptosis was reduced significantly with p38 and caspase-9 inhibitors. Induction of the endoplasmic reticulum stress response by anoxia/recovery or tunicamycin (monitored by induction of Bip or Grp94 expression, phosphorylation of eukaryotic translation initiation factor 2alpha subunit, expression of CHOP, and activation of caspase-12) was attenuated in cells that overexpress SLK. Thus, SLK is an anoxia/recovery-dependent kinase that is activated via homodimerization and that signals via ASK1 and p38 to promote apoptosis. Attenuation of the protective aspects of the endoplasmic reticulum stress response by SLK may contribute to its proapoptotic effect.