Effects of glucose 6-phosphate and hemin on activation of heme-regulated eIF-2 alpha kinase in gel-filtered reticulocyte lysates

In heme-deficient reticulocyte lysates, protein synthesis initiation is inhibited due to the activation of a heme-regulated protein kinase which blocks protein synthesis by the specific phosphorylation of the alpha-sub-unit of eukaryotic initiation factor 2 (eIF-2 alpha). The restoration of synthesis requires both hemin and glucose-6-P (Ernst, V., Levin, D. H., and London, I. M. (1978) J. Biol. Chem. 253, 7163-7172). The sugar phosphate fulfills two functions in initiation: (i) the generation of NADPH, and (ii) an effector function in some step in initiation. This latter effect is readily demonstrated in lysates depleted of low molecular weight components by filtration in dextran gels. In gel-filtered lysates, linear protein synthesis is sustained only by the addition of both hemin (20 microM) and glucose-6-P (or 2-deoxyglucose-6-P) (50-500 microM). The omission of either component gives rise to inhibitions which are characterized by the activation of heme-regulated eIF-2 alpha kinase and the concomitant phosphorylation of both endogenous heme-regulated eIF-2 alpha kinase and endogenous eIF-2 alpha, indicating that glucose-6-P is involved in the regulation of heme-regulated eIF-2 alpha kinase. In support of this, we find (a) that gel-filtered lysates incubated with hemin but depleted of glucose-6-P produce sufficient heme-regulated eIF-2 alpha kinase to inhibit protein synthesis when mixed with normal hemin-supplemented lysates; (b) the inhibitions of protein synthesis produced by heme-regulated eIF-2 alpha kinase generated either in glucose-6-P-depleted lysates or heme-deficient lysates are reversed by added eIF-2; and (c) the eIF-2 alpha kinase activities formed in the absence of either hemin or glucose-6-P are both neutralized by an anti-heme-regulated eIF-2 alpha kinase antiserum. We conclude that the physiological activation of heme-regulated eIF-2 alpha kinase is controlled by both hemin and glucose-6-P.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

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.     

Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization

It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.     

Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.     

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.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity

Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.     

A novel human gene similar to the protein kinase (PK) coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J. , Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn(2+)-dependent serine-threonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys(113) within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.     

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.     

Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue

In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.     

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.     

Sphinganine effects on chemoattractant-induced diacylglycerol generation, calcium fluxes, superoxide production, and on cell viability in the human neutrophil. Delivery of sphinganine with bovine serum albumin minimizes cytotoxicity without affecting inhibition of the respiratory burst

Long chain bases (sphinganine and sphingosine) are potent inhibitors of protein kinase C in an in vitro mixed micelle-reconstituted system (Hannun, Y. A., Loomis, C. R., Merrill, A. H. J., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609) and block activation of the superoxide-generating respiratory burst in human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). In the present studies, we have investigated the effects of sphinganine on cellular levels of the second messengers related to phosphoinositide turnover: diacylglycerol and calcium. We find that sphinganine added from a stock solution containing equimolar or greater bovine serum albumin had no effect on either formyl-methionyl-leucyl-phenylalanine-stimulated calcium fluxes or diacylglycerol generation, at levels which completely blocked activation of superoxide generation. In addition, there was no effect of sphinganine on cell viability in this concentration range. These data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action. When sphinganine was added from a stock in dimethyl sulfoxide, significant cytotoxic effects (assayed by trypan blue exclusion, release of cellular lactate dehydrogenase, and leakage of Quin2) were seen at concentrations nearer those which inhibited the respiratory burst. Cytotoxicity was inversely proportional to cell concentration and was probably due to detergent micelle formation which occurs in the absence of albumin. These studies emphasize the importance of the method of delivery and the consideration of cytotoxic effects, but indicate that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms.     

Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.