Phosphorylation of SAV1 by mammalian ste20-like kinase promotes cell death

The mammalian ste20-like kinase (MST) pathway is important in the regulation of apoptosis and cell cycle and emerges as a novel tumor suppressor pathway. MST-induced phosphorylation of Salvador homolog 1 (SAV1), which is a scaffold protein, has not been evaluated in detail. We performed a mass spectrometric analysis of the SAV1 protein that was co-expressed with MST2. Phosphorylation was detected at Thr-26, Ser-27, Ser-36 and Ser-269. Although single or double mutations had little effects, the mutation of all four residues in SAV1 to Ala (SAV1-4A) had inhibitory effects on the MST pathway. MST2-mediated induction of SAV1-4A protein levels, SAV1-4A interaction with MST2 and the self-dimerization of SAV1-4A were weaker compared to those of wild-type SAV1. SAV1-4A inhibited MST2- and K-RasG12V-induced cell death of MCF7 cells. These results suggest that MST-mediated phosphorylation of four residues within SAV1 may be important in the induction of cell death by the MST pathway.     

Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST).

Mammalian STE20-like kinase (MST) is a member of the yeast STE20-related kinase family and proteolytically activated by caspase during apoptosis. However, its other cellular functions are not known, including its activation mechanism, substrate(s), and subcellular localization. In this report, using anti-MST monoclonal antibodies, we clearly show that endogenous MST is localized in cytoplasm in a leptomycin B-dependent manner. Analyses with serial deletions and point mutations show that MST has two functional nuclear export signals and, unexpectedly, another localization motif for nuclear import. When cells are treated with leptomycin, monomeric MST is accumulated more rapidly in the nucleus than dimeric MST, indicating that dimerization contributes to the cytoplasmic retention of MST. Okadaic acid, an inhibitor of phosphatase 2A, induces activation of MST and translocation into the nucleus. Using phosphopeptide-specific antibody, we directly show that okadaic acid induces phosphorylation in the activation loop of MST, and, once phosphorylated, MST is rapidly translocated to the nucleus. However, kinase-deficient MST does not enter the nucleus, indicating that phosphorylation and activation is required for okadaic acid-induced nuclear translocation. In apoptotic cells, the activation of MST does not require phosphorylation in the activation loop and occurs through the release of C-terminal regulatory domain by caspase-dependent cleavage. Kinase-deficient MST functions dominant-negatively and represses okadaic acid-induced morphological change indicating that MST plays a role in okadaic acid-induced cellular shrinkage. Our identification of cytoplasmic and nuclear localization motifs and phosphorylation-dependent translocation of MST suggests that regulation of localization is important to the biological function of MST, including its effects on cellular morphology.     

The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway

The Salvador-Warts-Hippo (SWH) pathway is a complex signaling network that controls both developmental and regenerative tissue growth. Using a genetic screen in Drosophila melanogaster, we identified the sterile 20-like kinase, Tao-1, as an SWH pathway member. Tao-1 controls various biological phenomena, including microtubule dynamics, animal behavior, and brain development. Here we describe a role for Tao-1 as a regulator of epithelial tissue growth that modulates activity of the core SWH pathway kinase cassette. Tao-1 functions together with Hippo to activate Warts-mediated repression of Yorkie. Tao-1's ability to control SWH pathway activity is evolutionarily conserved because human TAO1 can suppress activity of the Yorkie ortholog, YAP. Human TAO1 controls SWH pathway activity by phosphorylating, and activating, the Hippo ortholog, MST2. Given that SWH pathway activity is subverted in many human cancers, our findings identify human TAO kinases as potential tumor suppressor genes.     

Phosphorylation of troponin I by protein kinase C: Mechanism of inhibition by calmodulin and troponin C

The mechanism by which calmodulin and troponin C influence phosphorylation of troponin I (TnI) by protein kinase C was investigated. The phosphorylation of TnI by protein kinase C requires the presence of acidic phospholipid, calcium and diacylglycerol. Light scattering intensity and fluorescence intensity experiments showed that TnI associated with the phospholipid membranes and caused extensive aggregation. In the presence of Ca²⁺, TnI-phospholipid interactions were prevented by approximately stoichiometric amounts of either troponin C or calmodulin. Troponin C was shown to completely inhibit phosphorylation of TnI by either protein kianse C or by phosphorylase b kinase. In contrast, calmodulin completely inhibited phosphorylation of TnI by protein kinase C, but had only little effect on TnI phosphorylation by phosphorylase b kinase. Inhibition by calmodulin did not appear to be due to interaction with PKC, since calmodulin mildly increased protein kinase C phosphorylation of histone III-S. The ratio of phosphoserine to phosphothreonine in protein kinase C-phosphorylated TnI remained approximately constant for reactions inhibited by up to 90% by clamodulin. TnI interactions with phospholipid and phosphorylation of TnI by PKC were also prevented by high salt concentrations. However, salt concentrations adequate to inhibit phosphorylation were sufficient to dissociate only TnI, but not protein kinase C from the membrane. These results suggest that the binding of TnI to phospholipid is required for phosphorylation by protein kinase C and that prevention of this binding by any means completely inhibited phosphorylation of TnI by protein kinase C.     

Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.     

Phosphorylation of troponin I by protein kinase C: mechanism of inhibition by calmodulin and troponin C

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

Inhibition of cell migration by autophosphorylated mammalian sterile 20-like kinase 3 (MST3) involves paxillin and protein-tyrosine phosphatase-PEST

MST3 is a member of the sterile-20 protein kinase family with a unique preference for manganese ion as a cofactor in vitro; however, its biological function is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF-7 cells with reduced expression of E-cadherin at the edge of migrating cells. The alteration of cellular migration and protruding can be rescued by RNA interference-resistant MST3. The expression of surface integrin and Golgi apparatus was not altered, but phosphorylation on tyrosine 118 and tyrosine 31 of paxillin was attenuated by MST3 small interfering RNA (siRNA). Threonine 178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro. Mutant T178A MST3, containing alanine instead of threonine at codon 178, lost autophosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in Madin-Darby canine kidney cells. MST3 could phosphorylate the protein-tyrosine phosphatase (PTP)-PEST and inhibit the tyrosine phosphatase activity of PTP-PEST. We conclude that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST.     

Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1.

Mst1 is a ubiquitously expressed serine-threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3-like activity during apoptosis induced by either cross-linking CD95/Fas or by staurosporine treatment. CD95/Fas-induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD-fmk, the more selective caspase inhibitor DEVD-CHO and by the viral serpin CrmA. Caspase-mediated cleavage of Mst1 removes the C-terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase-mediated cleavage activating Mst1. Overexpression of either wild-type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase-dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co-transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.     

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.     

Deletion of mammalian sterile 20-like kinase 1 attenuates neuronal loss and improves locomotor function in a mouse model of spinal cord trauma

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.     

Phosphorylation of cardiac troponin by guanosine 3':5'-monophosphate-dependent protein kinase.

Homogeneous cGMP-dependent protein kinase catalyzes the rapid incorporation of phosphate, specifically into the inhibitory subunit of purified cardiac troponin with a maximal incorporation of 1 mol of phosphate/mol of troponin. When troponin was incubated in the presence of both cGMP- and cAMP-dependent protein kinases, a maximal incorporation of 1 mol of phosphate/mol of troponin was observed which suggested phosphorylation of the same site by the two kinases. Both cyclic nucleotide-dependent kinases had similar Km values for troponin, but the Vmax value for the phosphorylation reaction catalyzed by cAMP-dependent protein kinase was 12-fold greater than the value obtained for cGMP-dependent protein kinase.     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.     

Phosphorylation of rabbit cardiac-muscle troponin I by phosphorylase kinase. The effect of adrenaline.

1. Incubation of rabbit cardiac-muscle troponin I with phosphorylase b kinase leads to the incorporation of .07-1.2 mol of Pi/mol. 2. The major site of phosphorylation is a serine residue at position 72. 3. Lesser amounts of phosphate are incorporated into threonine-138, threonine-162 and serine 20. 4. Serine-20 is the only site that contains a significant amount of phosphate before incubation with phosphorylase b kinase. 5. Unlike the situation with serine-20, the extent of phosphorylation of serine-72 and threonine-138 in the perfused rabbit heart does not change when the heart is exposed to adrenaline (4 microM).     

Phosphorylation and mutation of human cardiac troponin I deferentially destabilize the interaction of the functional regions of troponin I with troponin C

We have utilized 2D [(1)H,(15)N]HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC.3Ca(2+), T142 phosphorylation reduced the affinity of cIp for cCTnC.2Ca(2+) by approximately 14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC.Ca(2+) by approximately 10-fold. The mutation R144G caused an approximately 6-fold affinity decrease of cIp for cCTnC.2Ca(2+) and mutation R161W destabilized the interaction of cSp and cNTnC.Ca(2+) by approximately 1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC.2Ca(2+) was reduced approximately 19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC.Ca(2+) was reduced approximately 4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC.2Ca(2+), the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC.Ca(2+), demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.     

Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.     

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.