A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function

Recently it has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 (Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300). In cells expressing Bcl2, dephosphorylation of Bcl2 appears to be required for ceramide-induced cell death because treatment of cells with low doses of the PP2A inhibitor okadaic acid blocks Bcl2 dephosphorylation and promotes cell survival. Furthermore, the non-phosphorylatable (i.e. PP2A-resistant) gain-of-function S70E mutant Bcl2 can protect cells from ceramide-induced apoptosis. These findings support a model whereby Bcl2 function is regulated by PP2A. PP2A is a heterotrimer that contains a catalytic C-subunit, a structural A-subunit, and a regulatory B-subunit. The A- and C-subunits are fairly conserved and ubiquitously expressed, and they form the catalytic complex of the phosphatase. In contrast, there are at least three families of diverse B-subunit molecules that vary in expression temporally and by tissue type. It is hypothesized that ceramide regulates PP2A via the B-subunit. Thus, understanding the mechanism of how PP2A regulates Bcl2 phosphorylation status and how ceramide might regulate this process requires identification of the regulatory B-subunit of PP2A that comprises the Bcl2 phosphatase. Results indicate that the B56 alpha-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 alpha associates with Bcl2 as evidenced by pull-down experiments, (b) B56 alpha co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 alpha to mitochondrial membranes, and (d) overexpression of B56 alpha promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide. These findings suggest a role for B56 alpha in regulating the Bcl2 phosphatase.     

The structural requirements for ceramide activation of serine-threonine protein phosphatases

The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C(6) ceramide activated the catalytic subunit of PP2A (PP2Ac) approximately 3-fold in a stereospecific manner. In contrast, saturation of the 4-5 double bond, producing D-erythro-dihydro C(6) ceramide, inhibited PP2Ac (IC(50) = 8.5 microM). Furthermore, phyto C(6) ceramide, D-erythro-dehydro C(6) ceramide, and D-erythro-cis-C(6) ceramide had no effect on PP2Ac activity. Modification of the sphingoid chain also abolished the ability of ceramide to activate PP2Ac. Further studies demonstrated the requirement for the amide group, the primary hydroxyl group, and the secondary hydroxyl group of the sphingoid backbone for activation of PP2Ac through the synthesis and evaluation of D-erythro-urea C(6) ceramide, L-erythro-urea C(6) ceramide, D-erythro-N-methyl C(6) ceramide, D-erythro-L-O-methyl C(6) ceramide, D-erythro-3-O-methyl C(6) ceramide, and (2S) 3-keto C(6) ceramide. None of these compounds induced significant activation of PP2Ac. Liposome binding studies were also conducted using analogs of D-erythro-C C(6) ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP. This study demonstrates strict structural requirements for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP. The studies also begin to define features that transform ceramide analogs into inhibitors of CAPP.     

Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX

The sphingolipid ceramide is an important second signal molecule and potent apoptotic agent. The production of ceramide is associated with virtually every known stress stimulus, and thus, generation of this sphingolipid has been suggested as a universal feature of apoptosis. Recent studies suggest that an important component of cell death following diverse stress stimuli (e.g. interleukin-3 withdrawal, sodium arsenite treatment, and peroxide treatment) is the activation of the double-stranded RNA-activable protein kinase, PKR, resulting in the inhibition of protein synthesis (Ito, T., Jagus, R., and May, W. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7455-7459). The recently discovered cellular PKR activator, RAX, is phosphorylated in association with PKR activation (Ito, T., Yang, M., and May, W. S. (1999) J. Biol. Chem. 274, 15427-15432). Since RAX is phosphorylated by an as yet undetermined SAPK and ceramide is a potent activator of SAPKs such as JNK, a role for ceramide in the activation of RAX might be possible. Results indicate that overexpression of exogenous RAX potentiates ceramide-induced killing. Furthermore, ceramide can potently inhibit protein synthesis. Since ceramide potently promotes RAX and eukaryotic initiation factor-2alpha phosphorylation, a possible role for ceramide in this process may involve the activation of PKR by RAX. Since 2-aminopurine, a serine/threonine kinase inhibitor that has previously been shown to inhibit PKR, blocks both the potentiation of ceramide killing by RAX and ceramide-induced inhibition of protein synthesis, ceramide appears to promote PKR activation, at least indirectly. Collectively, these findings suggest a novel role for ceramide in the regulation of protein synthesis and apoptosis.     

An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line

An intracellular (ATP + Mg2+)-dependent Ca2+ pumping mechanism has been identified and characterized within the cultured clonal neuroblastoma cell line N1E-115. Using cell suspensions treated with 0.005% saponin which selectively permeabilizes the plasma membrane in 95-98% of the cells, it was possible to show clearly that the intracellular Ca2+ pump mechanism is of non-plasma membrane origin and therefore can be compared directly with the Ca2+ pump characterized in detail in synaptosomal membrane vesicles (Gill, D. L., Grollman, E. F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192; Gill, D. L., Chueh, S. H., and Whitlow, C. L. (1984) J. Biol. Chem. 259, 10807-10813) which was proven by flux reversal studies to be derived from the neural plasma membrane (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990). The intracellular Ca2+ pump in N1E-115 cells is distinct from mitochondrial Ca2+ accumulation and is increased up to 8-fold higher as cells reach confluency. In similarity to the neural plasma membrane pump, the intracellular Ca2+ pump within N1E-115 cells has high affinity for Ca2+ (Km = 0.28 microM), is dependent on both ATP (Km = 26 microM) and either Mg2+ or Mn2+ which half-maximally activate Ca2+ pumping at 0.35 mM and 0.32 mM, respectively, and shows similar specificity for Sr2+ and Ba2+ which half-maximally inhibit Ca2+ transport at 50 microM and 1.5 mM, respectively. In contrast to the neural plasma membrane pump, the intracellular Ca2+ pump displays approximately 40-fold higher sensitivity to La3+ (IC50 = 5 microM) and an apparent 400-fold lower sensitivity to VO4(3-) (IC50 = 185 microM), although the inhibitory effectiveness of VO4(3-) is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO4(3-) in the absence of ATP (apparent IC50 = 5 microM). In further contrast to the neural plasma membrane Ca2+ pump, the intracellular pump within N1E-115 cells is stimulated more than 20-fold by oxalate (giving prolonged linear Ca2+ accumulation), is resistant to low saponin concentrations, and is not modified by calmodulin even after extensive treatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and/or calmodulin antagonist drugs. However, calmidazolium is effective in inhibiting the intracellular Ca2+ pump with an IC50 of approximately 2 microM.     

Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid.

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases, which include protein phosphatase-2A (PP2A) and protein phosphatase-1 (PP1) with roles in regulating apoptosis and cell growth. Thus far, in vitro studies on ceramide-activated protein phosphatases have been restricted to the use of short chain ceramides, limiting the extent of mechanistic insight. In this study, we show that the long chain D-erythro-C18-ceramide activated PP2A (AB'C trimer), PP2Ac (catalytic subunit of PP2A), and PP1gammac and -alphac (catalytic subunits of PP1gamma and -1alpha isoforms, respectively) 2-6-fold in the presence of dodecane, a lipid-solubilizing agent, with 50% maximal activation achieved at approximately 10 microM D-erythro-C18-ceramide. The diastereoisomers of D-erythroC18-ceramide, D-threo-, and L-threo-C18-ceramide, as well as the enantiomeric L-erythro-C18-ceramide, did not activate PP1 or PP2A, but they inhibited PP1 and PP2A activity. The addition of phosphatidic acid decreased the basal activity of PP1c but also increased the stimulation by D-erythro-C18-ceramide from 1.8- to 2. 8-fold and decreased the EC50 of D-erythro-C18-ceramide to 4.45 microM. The addition of 150 mM KCl decreased the basal activity of PP1 and the dose of D-erythro-C18-ceramide necessary to activate PP1c (EC50 = 6.25 microM) and increased the ceramide responsiveness up to 10-17-fold. These studies disclose stereospecific activation of PP1 and PP2A by long chain natural ceramides under near physiologic ionic strengths in vitro. The implications of these studies for mechanisms of ceramide action are discussed.     

Cantharimides: a new class of modified cantharidin analogues inhibiting protein phosphatases 1 and 2A.

Cantharidin and its analogues have been of considerable interest as potent inhibitors of the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A). However, limited modifications to the parent compounds is tolerated. As part of an on-going study we have developed a new series of cantharidin analogues, the cantharimides. Inhibition studies indicate that cantharimides possessing a D- or L-histidine, are more potent inhibitors of PP1 and PP2A (PP1 IC(50)=3.22+/-0.7 microM; PP2A IC(50)=0.81+/-0.1 microM and PP1 IC(50)=2.82+/-0.6 microM; PP2A IC(50)=1.35+/-0.3 microM, respectively) than norcantharidin (PP1 IC(50)=5.31+/-0.76 microM; PP2A IC(50)=2.9+/-1.04 microM) and essentially equipotent with cantharidin (PP1 IC(50)=3.6+/-0.42 microM; PP2A IC(50)=0.36+/-0.08 microM). Cantharimides with non-polar or acidic amino acid residues are only poor inhibitors of PP1 and PP2A.     

Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis.

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.     

OSBPa,a predicted oxysterol binding protein of Dictyostelium,is required for regulated entry into culmination

In our previous papers [e.g. Sato et al., J. Biol. Chem. 273 (1998) 21455-21462], we have shown that prodigiosins can uncouple various H(+)-ATPases through their H(+)/Cl(-) symport activity. BE-18591 is an enamine of 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (tambjamine group antibiotics) which resembles the prodigiosins. We found that BE-18591 was a new group of antibiotics that uncouples various H(+)-ATPases: it inhibited proton pump activities with IC(50)s of about 1-2 nM (about 20 pmol/mg protein) for submitochondrial particles as well as gastric vesicles and of 230 nM (about 230 pmol/mg protein) for lysosomes, but it had little effect on their ATP hydrolyses (up to 10 microM), a property of H(+)/Cl(-) symport activity. At low concentrations (<1 microM), BE-18591 inhibited immunoproliferation, the IC(50) of lipopolysaccharide-stimulated mouse splenocytes was 38 nM, that of Concanavalin A-stimulated cells was 230 nM. Gastritis of rabbits was also inhibited. At higher concentrations (>1 microM), BE-18591 induced neurite outgrowth (15% induction in 48 h at 4 microM), inhibited bone resorption (approximately 35% in 48 h at 10 microM) and caused cell death (approximately 30% in 48 h at 4 microM) but with little apoptosis.     

Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.     

Heterocyclic substituted cantharidin and norcantharidin analogues--synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity

Norcantharidin (3) is a potent PP1 (IC(50)=9.0+/-1.4 microM) and PP2A (IC(50)=3.0+/-0.4 microM) inhibitor with 3-fold PP2A selectivity and induces growth inhibition (GI(50) approximately 45 microM) across a range of human cancer cell lines including those of colorectal (HT29, SW480), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (DU145), neuroblastoma (BE2-C), and glioblastoma (SJ-G2) origin. Until now limited modifications to the parent compound have been tolerated. Surprisingly, simple heterocyclic half-acid norcantharidin analogues are more active than the original lead compound, with the morphilino-substituted (9) being a more potent (IC(50)=2.8+/-0.10 microM) and selective (4.6-fold) PP2A inhibitor with greater in vitro cytotoxicity (GI(50) approximately 9.6 microM) relative to norcantharidin. The analogous thiomorpholine-substituted (10) displays increased PP1 inhibition (IC(50)=3.2+/-0 microM) and reduced PP2A inhibition (IC(50)=5.1+/-0.41 microM), to norcantharidin. Synthesis of the analogous cantharidin analogue (19) with incorporation of the amine nitrogen into the heterocycle further increases PP1 (IC(50)=5.9+/-2.2 microM) and PP2A (IC(50)=0.79+/-0.1 microM) inhibition and cell cytotoxicity (GI(50) approximately 3.3 microM). These analogues represent the most potent cantharidin analogues thus reported.     

Phosphatidic acid is a potent and selective inhibitor of protein phosphatase 1 and an inhibitor of ceramide-mediated responses.

In the present study, we report that phosphatidic acid (PA) functions as a novel, potent, and selective inhibitor of protein phosphatase 1 (PP1). The catalytic subunit of PP1alpha was inhibited by PA dose-dependently in a noncompetitive manner with a K(i) value of 80 nM. The inhibition by PA was specific to PP1 as PA failed to inhibit protein phosphatase 2A (PP2A) or PP2B. Furthermore, PA was the most effective and potent inhibitor of PP1 compared with other phospholipids. Because we recently showed that ceramides activated PP1, we next examined the effects of PA on ceramide stimulation of PP1. PA inhibited both basal and ceramide-stimulated PP1 activities, and ceramide showed potent and stereoselective activation of PP1 in the presence of PA. Next, the effects of PA on ceramide-induced responses were examined. Molt-4 cells took up PA dose- and time-dependently such that by 1 and 3 h, uptake of PA was 0.37 and 0. 65% of total PA added, respectively. PA at 30 microM and calyculin A at 10 nM (an inhibitor of PP1 and PP2A at low concentrations), but not okadaic acid at 10 nM (a PP2A inhibitor at low concentrations) prevented poly(ADP-ribose) polymerase proteolysis induced by C(6)-ceramide. Moreover, the combination of PA with okadaic acid prevented retinoblastoma gene product dephosphorylation induced by C(6)-ceramide. These data suggest that PA functions as a specific regulator of PP1 and may reverse or counteract those effects of ceramide that are mediated by PP1, such as apoptosis and retinoblastoma gene product dephosphorylation.     

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively enriched in PP1gamma(1) over PP1beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H. , Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homologous neurabin residues 436-479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1gamma(1) from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1gamma(1) over PP1beta. Moreover, immunoprecipitation of PP1gamma(1) from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.     

Mechanisms of mastoparan-stimulated surfactant secretion from isolated pulmonary alveolar type 2 cells.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.     

Modified norcantharidins; synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC(50)=13+/-5 microM; PP2A IC(50)=7+/-3 microM) and 16 (PP1 IC(50)=18+/-8 microM; PP2A IC(50)=3.2+/-0.4 microM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).     

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella coupled with ATP hydrolysis

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella in relation to ATP hydrolysis was examined by tryptic digestion. Tryptic digestion of the beta chain in the presence of 2 mM ATP (ADP) and 100 microM vanadate (Vi) or in the presence of 4 mM ATP gamma S produced different polypeptides from in the case of no addition. The difference was similar to the result previously reported for 21S outer-arm dynein heavy chains [Inaba, K. & Mohri, H. (1989) J. Biol. Chem. 264, 8384-8388]. Unlike the tryptic digestion pattern of 21S dynein heavy chains, however, the 135-kDa polypeptide was consistently produced from the beta chain, even in the presence of ATP (ADP) and Vi. The tryptic digestion pattern of the 21S particle reconstituted from the separated a chain, the beta/IC1 complex and the IC2/IC3 complex [Tang, W.-J.Y., Bell, C.W., Sale, W.S., & Gibbons, I.R. (1982) J. Biol. Chem. 257, 508-515] was similar to that of intact 21S dynein; the 135-kDa polypeptide was only slightly produced in the presence of ATP and Vi. The digestion rate constant of the 135-kDa polypeptide from the beta chain in the presence of ATP and Vi was significantly decreased as compared with in the case of 21S dynein or that of the reconstituted 21S particle. These results suggest that the trypsin sensitivity of the 135-kDa region of the beta chain changes with the association of the beta/ICI complex with the alpha chain and the IC2/IC3 complex in the presence of ATP and Vi.     

Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101–11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.     

Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering.

Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.     

Membrane restructuring via ceramide results in enhanced solute efflux.

The capacity of ceramides to modify the permeability barrier of cell membranes has been explored. Membrane efflux induced either by in situ generated ceramides (through enzymatic cleavage of sphingomyelin) or by addition of ceramides to preformed membranes has been studied. Large unilamellar vesicles composed of different phospholipids and cholesterol, and containing entrapped fluorescent molecules, have been used as a system to assay ceramide-dependent efflux. Small proportions of ceramide (10 mol % of total lipid) that may exist under physiological conditions of ceramide-dependent signaling have been used in most experiments. When long chain (egg-derived) ceramides are used, both externally added or enzymatically produced ceramides induce release of vesicle contents. However, the same proportion of ceramides generated by sphingomyelinase induce faster and more extensive efflux than when added in organic solution to the preformed vesicles. Under our conditions 10 mol % of N-acetylsphingosine (C(2)-ceramide) did not induce any efflux. On the other hand, sphingomyelinase treatment of bilayers containing 50 mol % sphingomyelin gave rise to release of fluorescein-derivatised dextrans of molecular mass approximately 20 kDa, i.e. larger than cytochrome c. These results have been discussed in the light of our own previous data (Ruiz-Argüello, M. B., Basa?ez, G., Go?i, F. M., and Alonso, A. (1996) J. Biol. Chem. 271, 26616-26621) and of the observations by Siskind and Colombini (Siskind, L. J., and Colombini, M. (2000) J. Biol. Chem. 275, 38640-38644). Our spectroscopic observations appear to be in good agreement with the electrophysiological studies of the latter authors. Furthermore, some experiments in this paper have been designed to explore the mechanism of ceramide-induced efflux. Two properties of ceramide, namely its capacity to induce negative monolayer curvature and its tendency to segregate into ceramide-rich domains, appear to be important in the membrane restructuring process.     

Identification and characterization of protein phosphatase 2C activation by ceramide

Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.