Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A.

Phosphorylation of Bcl2 at serine 70 is required for its potent anti-apoptotic function. We have recently shown that Bcl2 phosphorylation is a dynamic process that involves the protein kinase C alpha and protein phosphatase 2A (PP2A) (Ruvolo, P. P., Deng, X., Carr, B. K., and May, W. S. (1998) J. Biol. Chem. 273, 25436-25442; and Deng, X., Ito, T., Carr, B. K., Mumby, M. C., and May, W. S. (1998) J. Biol. Chem. 273, 34157-34163). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2-dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain-of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC50 > 50 microM). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC50 = 14 microM). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.     

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.     

Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.     

A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells

Our previous study suggested that N,N-dimethylsphingosine, but not unsubstituted sphingosine, could be a modulator of protein kinase C in epidermoid carcinoma A431 cells, since N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial sphingosine preparation has an enhancing effect on epidermal growth factor (EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of N,N-dimethylsphingosine as compared with lyso-glycosphingolipids and other sphingolipid breakdown products on EGF receptor autophosphorylation and (ii) demonstration of endogenous N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted sphingosine in A431 cells. The autophosphorylation of EGF receptor in the absence of detergent was strongly enhanced by N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of EGF and synergistic in the presence of EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-glycosphingolipids tested. Labeling of sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of sphingolipids by standard procedures, resulted in clear bands corresponding to N,N-dimethylsphingosine and ceramide, whereas the band corresponding to sphingosine was virtually absent. The bands corresponding to N,N-dimethylsphingosine and ceramide intensified when cells were treated with metabolic inhibitor for UDP-Glc:Cer beta-Glc transferase (which causes accumulation of ceramide). These results indicate that N,N-dimethylsphingosine acts as a stereospecific enhancer for EGF receptor kinase and is able to produce EGF-like activity in vitro even in the absence of EGF and detergent. Under physiological conditions, N,N-dimethylsphingosine is the major catabolite resulting from ceramide breakdown.     

Activation of acid sphingomyelinase by protein kinase Cdelta-mediated phosphorylation

Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.     

Palmitoylation of cysteine 69 from the COOH-terminal of band 3 protein in the human erythrocyte membrane. Acylation occurs in the middle of the consensus sequence of F--I-IICLAVL found in band 3 protein and G2 protein of Rift Valley fever virus

One of the major physiologic functions of erythrocytes is the mediation of chloride-bicarbonate exchange in the transport of carbon dioxide from the tissues to the lungs. The anion exchange is mediated by a typical polytopic transmembrane protein in the cell membrane, designated Band 3. A carboxyl-terminal peptide of Band 3 was affinity-labeled with pyridoxal phosphate, a substrate for the anion transport system, and then sequenced (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The 10th amino acid residue of the peptide could not be determined, suggesting post-translational modification of the residue. In the present communication, we have investigated the molecular structure of human Band 3 and the COOH-terminal 8500-dalton peptide using gas-liquid chromatography-mass spectrometry. Band 3 was modified covalently by fatty acids and these acids were released from Band 3 by hydroxylamine treatment at either pH 7 or 11, indicating that the linkage between Band 3 and the fatty acid is a thio ester bond. 1 mol of Band 3 interacted with 1 mol of fatty acid at a cysteine residue located 69 residues from the COOH terminus of Band 3. The fatty acids used in the modification were myristate, palmitate, oleate, and stearate, with palmitate being the major component. The esterified site is close to the site affinity-labeled with pyridoxal phosphate (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The amino acid sequence including the acylation site was Phe-Thr-Gly-Ile-Gln-Ile-Ile-Cys-Leu-Ala-Val-Leu, which is conserved in the G2 protein of Rift Valley fever virus as Phe-Ser-Ser-Ile-Ala-Ile-Ile-Cys-Leu-Ala-Val-Leu. The G2 protein, like Band 3, is a polytopic transmembrane protein. Although acylation of the cysteine residue of G2 protein has not been examined, the Phe-X-X-Ile-X-Ile-Ile-Cys-Leu-Ala-Val-Leu sequence could be a common motif for fatty acylation of certain membrane proteins.     

Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene

D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.     

A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL

Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.     

Tic40, a new "old" subunit of the chloroplast protein import translocon

The protein import translocon at the inner envelope of chloroplasts (Tic complex) is a heteroligomeric multisubunit complex. Here, we describe Tic40 from pea as a new component of this complex. Tic40 from pea is a homologue of a protein described earlier from Brassica napus as Cim/Com44 or the Toc36 subunit of the translocon at the outer envelope of chloroplasts, respectively (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264-32271; Ko, K., Budd, D., Wu, C., Seibert, F., Kourtz, L., and Ko, Z. W. (1995) J. Biol. Chem. 270, 28601-28608; Pang, P., Meathrel, K., and Ko, K. (1997) J. Biol. Chem. 272, 25623-25627). Tic40 can be covalently connected to Tic110 by the formation of a disulfide bridge under oxidizing conditions, indicating its close physical proximity to an established translocon component. The Tic40 protein is synthesized in the cytosol as a precursor with an N-terminal cleavable chloroplast targeting signal and imported into the organelle via the general import pathway. Immunoblotting and immunogold-labeling studies exclusively confine Tic40 to the chloroplastic inner envelope, in which it is anchored by a single putative transmembrane span.     

Direct photoaffinity labeling of junctional sarcoplasmic reticulum with [14C]doxorubicin

Doxorubicin, an anticancer drug, induces Ca2+ release from the terminal cisternae (TC) of skeletal muscle (Zorzato, F., Salviati, G., Facchinetti, T., and Volpe, P. (1985) J. Biol. Chem. 260, 7349-7355). Long wave ultraviolet irradiation of a TC fraction with morphologically intact feet structures (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) in the presence of [14C]doxorubicin, led to covalent photolabeling of two proteins that exhibited apparent Mr values of 350,000 and 170,000. Such proteins were found to be absent in a fraction of longitudinal sarcoplasmic reticulum but enriched in junctional face membranes obtained by Triton X-100 treatment of the TC fraction. Three additional proteins with Mr values of 80,000, 60,000, and 30,000 were also faintly labeled in the junctional face membrane fraction. On a molar basis the highest level of incorporation was found in the 170,000-Da protein, probably a Ca2+-binding protein (Campbell, K. P., MacLennan, D. H., and Jorgensen, A. O. (1983) J. Biol. Chem. 258, 11267-11273). A lower level of labeling was observed in the 350,000-Da protein, tentatively identified as a component of the feet structures (Cadwell, J. J. S., and Caswell, A. H. (1982) J. Cell Biol. 93, 543-550). Photolabeling of junctional TC proteins did not occur if a 10-50-fold excess cold doxorubicin was included in the assay medium, indicating that it was displaceable and specific, and if ultraviolet irradiation was omitted. Photolabeling was inhibited by caffeine or ruthenium red, i.e. by an activator and an inhibitor of Ca2+ release from TC, respectively. Furthermore, photolabeling was prevented by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid suggesting that doxorubicin binding is Ca2+-dependent. Doxorubicin-binding proteins are constituents of the junctional sarcoplasmic reticulum and might be involved in modulating Ca2+ release from TC.     

Interaction between the chemotactic cAMP receptor and a detergent-insoluble membrane residue of Dictyostelium discoideum. Modulation by guanine nucleotides

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.     

Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71

p-Fluorosulfonylbenzoyl 5'-adenosine (FSO2BzAdo) was shown previously to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase II from porcine skeletal muscle (Zoller, M. J., and Taylor, S. S. (1979) J. Biol. Chem. 254, 8363-8368). The catalytic subunit of porcine heart cAMP-dependent protein kinase was also inhibited following incubation with FSO2[14C]BzAdo, and inhibition was shown to result from the stoichiometric, covalent modification of a single lysine residue. The amino acid sequence in an extended region around the carboxybenzenesulfonyl lysine (CBS-lysine) was elucidated by characterizing both tryptic and cyanogen bromide peptides containing the 14C-modified residue. The sequence in this region was Leu-Val-Lys-His-Lys-Glu-Thr-Gly-Asn-His-Phe-Ala-Met-Lys(CBS)-Ile-Leu-Asp-Lys-Glu-Lys-Val-Val-Lys-Leu-Lys-Gln-Ile. The covalently modified residue corresponded to lysine 71 in the overall polypeptide chain. Homologies to bovine heart catalytic subunit and to a site modified by FSO2BzAdo in phosphofructokinase are considered.     

Sphingosine-1-phosphate phosphohydrolase regulates endoplasmic reticulum-to-golgi trafficking of ceramide

Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4 degrees C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.     

Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance.

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases

The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6-phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo-2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP-dependent protein kinase, the synthetic peptide corresponding to the cAMP-dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP-dependent protein kinase.     

Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein

Particulate preparations from insulin-treated 3T3-L1 cells retain the enhanced ability to incorporate 32P from [gamma-32P]ATP into ribosomal protein S6 (Smith, C. J., Rubin, C. S., and Rosen, O. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2641-2645). A cyclic AMP-independent protein kinase that phosphorylates S6 and casein and that may be involved in the increase in S6 phosphorylation produced by insulin has been isolated based upon the observation that there is 1.5-3.0-fold higher activity in particulate preparations derived from insulin-treated cells than there is in comparable preparations from control cells. The enzyme activity was purified 2071-fold by KCl extraction, phosphocellulose chromatography, and gel filtration. The S6 phosphorylating activity was also characterized by its behavior on casein-Sepharose and DEAE-cellulose chromatography and its sedimentation in glycerol gradients. None of these procedures resolved the S6 and casein kinase activities. Some of the properties of this kinase, including a molecular weight of about 35,000, inhibition by F- or phosphate, chromatography on DEAE-cellulose and phosphocellulose, and insensitivity to inhibition by GTP, are similar to those of a previously described enzyme, casein kinase I (Dahmus, M. E. (1981) J. Biol. Chem. 256, 3319-3325; Hathaway, G. M., and Traugh, J. A. (1979) J. Biol. Chem. 254, 762-768).     

Anthranilate synthetase component II from Pseudomonas putida. Covalent structure and identification of the cysteine residue involved in catalysis

The complete amino acid sequence of carboxamidomethylated anthranilate synthetase component II (AS II) from Pseudomonas putida has been determined by analysis of cyanogen bromide fragments, tryptic peptides from the citraconylated protein, and by analysis of subdigests of these peptides. AS II is a single polypeptide chain of 197 residues having a calculated molecular weight of 21,684. Previous studies (Goto, Y., Keim, P. S., Zalkin, H., and Heinrikson, R. L. (1976) J. Biol. Chem, 251, 941-949) identified a cysteine residue required for the formation of an acyl-enzyme intermediate. The protein has 3 cysteine residues at positions 54, 79, and 140. Cysteine-79 was alkylated selectively by iodoacetamide and by the glutamine affinity analogue L-2-amino-4-oxo-5-chloropentanoic acid. Based on this evidence cysteine-79 is the active site residue involved in formation of the acyl-enzyme intermediate. Comparison of the P. putida AS II sequence with that of the NH2-terminal 60 residues of the enzyme from Escherichia coli shows 38% sequence identity.