Poly(ADP-ribose) modulates the properties of MARCKS proteins

In mammalian cells, the formation of DNA strand breaks is accompanied by synthesis of poly(ADP-ribose). This nucleic acid-like homopolymer may modulate protein functions by covalent and/or noncovalent interactions. Here we show that poly(ADP-ribose) binds strongly to the proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family, MARCKS and MARCKS-related protein (also MacMARCKS or F52). MARCKS proteins are myristoylated proteins associated with membranes and the actin cytoskeleton. As targets for both protein kinase C (PKC) and calmodulin (CaM), MARCKS proteins are thought to mediate cross-talk between these two signal transduction pathways. Dot blot assays show that poly(ADP-ribose) binds to MARCKS proteins at the highly basic effector domain. Complex formation between MARCKS-related protein and CaM as well as phosphorylation of MARCKS-related protein by the catalytic subunit of PKC are strongly inhibited by equimolar amounts of poly(ADP-ribose), suggesting a high affinity of poly(ADP-ribose) for MARCKS-related protein. Binding of MARCKS-related protein to membranes is also inhibited by poly(ADP-ribose). Finally, poly(ADP-ribose) efficiently reverses the actin-filament bundling activity of a peptide corresponding to the effector domain and inhibits the formation of actin filaments in vitro. Our results suggest that MARCKS proteins and actin could be targets of the poly(ADP-ribose) DNA damage signal pathway.     

Performance status and comorbidity in elderly cancer patients compared with young patients with neoplasia and elderly patients without neoplastic conditions

MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously analyzed MARCKS purified from bovine brain using capillary liquid chromatography/electrospray mass spectrometry and found that the protein structure differed significantly from that deduced from cDNA sequences [Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302]. Moreover, the alignment of the protein from various species showed a lack of any conserved sequences in the C-terminal half of the molecule. This prompted us to reexamine the C-terminal amino-acid sequence of bovine MARCKS. The purified protein was digested with lysyl endoprotease, and the obtained C-terminal peptide was further digested with either Staphylococcus V8 protease or NTCB. The small peptides thus obtained were analyzed by liquid chromatography/electrospray/tandem mass spectrometry. This combined with gas-phase Edman sequencing allowed us to determine the C-terminal primary structure. The sequence obtained differed significantly from that reported previously, and the comparison with other species revealed the presence of a novel conserved domain in the C-terminal region of MARCKS.     

Identification and characterization of cathepsin B as the cellular MARCKS cleaving enzyme

The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH4Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 x g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time- and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.     

The in vivo metabolism of 14C-glucose and 14C-glycine in insulin-treated northern pike (Esox lucius L.)

Component a of the erythrocyte membrane is a specific substrate for endogenous protein kinase activity and its phosphorylation is significantly decreased under assay conditions in myotonic muscular dystrophy (Roses, A.D., and Appel, S.H.J. Membr. Biol 20:51-58 (1975)). We have demonstrated substrate heterogeneity of two fractions of component a separated by concanavalin A (Con-A) sepharose chromatography. The fraction of component a that is retarded by Con A and eluted with alpha-methyl-D-glucoside does not accept the transfer of phosphate from [gamma-32 P] ATP as a substrate for endogenous protein kinase activity. The nonretarded fraction contains greater than 90% of the radioactive label. These experiments also confirm the carbohydrate heterogeneity of component a (Findley, J.B.C., J. Biol. Chem. 249:4398 (1974).     

The myristoyl moiety of myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein is embedded in the membrane

Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.     

Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.     

Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as a proteinase-adhesin polyprotein

The identification of proteinases of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications in the study of host-pathogen interactions as well as in the discovery of potential therapeutic and immunoprophylactic agents. We have cloned and characterized a gene that encodes the 50-kDa cysteine proteinase gingipain or Arg-gingipain-1 (RGP-1) described previously (Chen, Z., Potempa, J., Polanowski, A., Wikstrom, M., and Travis, J. (1992) J. Biol. Chem. 267, 18896-18901). Analysis of the amino acid sequence of RGP-1 deduced from the cloned DNA sequence showed that the biosynthesis of this proteinase involves processing of a polyprotein that contains multiple adhesin molecules located at its carboxyl terminus. This finding corroborates previous evidence (Pike R., McGraw, W., Potempa, J., and Travis, J. (1994) J. Biol. Chem. 269, 406-411) that RGP-1 is closely associated with adhesin molecules, and that high molecular weight forms of the proteinase are involved in the binding of erythrocytes.     

Molecular cloning and characterization of Porphyromonas gingivalis lysine-specific gingipain. A new member of an emerging family of pathogenic bacterial cysteine proteinases

The proteinases of Porphyromonas gingivalis are key virulence factors in the etiology and progression of periodontal disease. Previous work in our laboratories resulted in the purification of arginine- and lysine-specific cysteine proteinases, designated gingipains, that consist of several tightly associated protein subunits. Recent characterization of arginine-specific gingipain-1 (gingipain R1; RGP-1) revealed that the sequence is unique and that the protein subunits are initially translated as a polyprotein encoding a proteinase domain and multiple adhesin domains (Pavloff, N., Potempa, J., Pike, R. N., Prochazka, V., Kiefer, M. C., Travis, J., and Barr, P. J. (1995) J. Biol. Chem. 270, 1007-1010). We now show that the lysine-specific gingipain (gingipain K; KGP) is also biosynthesized as a polyprotein precursor that contains a proteinase domain that is 22% homologous to the proteinase domain of RGP-1 and multiple adhesin domains. This precursor is similarly processed at distinct sites to yield active KGP. The key catalytic residues in the proteinase domain of KGP are identical to those found in RGP-1, but there are significant differences elsewhere within this domain that likely contribute to the altered substrate specificity of KGP. Independent expression of the proteinase domain in insect cells has shown that KGP does not require the presence of the adhesin domains for correct folding to confer proteolytic activity.     

Residues 21-30 within the extracellular N-terminal region of the C5a receptor represent a binding domain for the C5a anaphylatoxin

The functions of the C5a anaphylatoxin are expressed through its interaction with a cell-surface receptor with seven transmembrane helices. The interaction of C5a with the receptor has been explained by a two-site model whereby recognition and effector sites on C5a bind, respectively, to recognition and effector domains on the receptor, leading to receptor activation (Chenoweth, D. E., and Hugli, T. E. (1980) Mol. Immunol. 17, 151-161. In addition, the extracellular N-terminal region of the C5a receptor has been implicated as the recognition domain for C5a, responsible for approximately 50% of the binding energy of the C5a-receptor complex (Mery, L., and Boulay, F. (1994) J. Biol. Chem. 269, 3457-3463; DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol. Chem. 269, 14446-14450). In this work, the interactions of C5a with the N-terminal domain of the C5a receptor were examined by use of recombinant human C5a molecules and peptide fragments M1NSFN5YTTPD10YGHYD15DKDTL20DLNTP25VDKTS30NTLR(hC5aRF-1-34), acetyl-HYD15DKDTL20DLNTP25VDKTS30NTLR (hC5aRF-13-34), and acetyl-TL20DLNTP25VDKTS30N-amide (hC5aRF-19-31) derived from human C5a receptor. Binding induced resonance perturbations in the NMR spectra of the receptor fragments and the C5a molecules indicated that the isolated Nterminal domain or residues 1-34 of the C5a receptor retain specific binding to C5a and to a C5a analog devoid of the agonistic C-terminal tail in the intact C5a. Residues of C5a perturbed by the binding of the receptor peptides are localized within the helical core of the C5a structure, in agreement with the results from functional studies employing mutated C5a and intact receptor molecules. All three receptor peptides, hC5aRF-1-34, hC5aRF-13-34, and hC5aRF-19-31, responded to the binding of C5a through the 21-30 region containing either hydrophobic, polar, or positively charged residues such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30. The 21-30 segment of all three receptor fragments was found to have a partially folded conformation in solution, independent of residues 1-18. These results indicate that a short peptide sequence, or residues 21-30, of the C5a receptor N terminus may constitute the binding domain for the recognition site on C5a.     

Interaction of the effector domain of MARCKS and MARCKS-related protein with lipid membranes revealed by electric potential measurements

We have investigated the binding of the effector domains of myristoylated alanine-rich C kinase substrate (MARCKS) and of MARCKS-related protein (MRP) to lipid model membranes. For membrane systems we used lipid monolayers on a Langmuir trough and black lipid membranes (BLM). The binding of the peptides was detected by monitoring changes in the boundary potential of the lipid membranes. The vibrating plate technique (VPT) and the method of inner field compensation (IFC) were used for the monolayer and for the BLM, respectively. We could show that the effector domain of MARCKS binds to acidic lipid membranes mainly via electrostatic interactions and to zwitterionic lipid membranes via hydrophobic interactions. Isobaric measurements on lipid monolayers revealed that binding of both effector domains is accompanied by partial insertion of the peptides into the membrane. Adsorption and insertion of the peptides could be followed simultaneously by the VPT and by recording the increase in area of the lipid monolayer, respectively. No temporal delay could be observed between adsorption and insertion of the peptides, demonstrating that adsorption is the rate-limiting step and that insertion is faster than the time resolution of the experiments, i.e., a few seconds. Both the IFC and the VPT did not show any significant difference between the behaviors of the effector domains of MARCKS and MRP. With the IFC we show that calcium can regulate the translocation of the MARCKS effector peptide between the membrane and calmodulin (CaM) in the bulk. Our results indicate, that the IFC and VPT are suitable qualitatively, and to a certain extent quantitatively, as membrane binding assays.     

Sustained activation of phospholipase D via adenosine A3 receptors is associated with enhancement of antigen- and Ca(2+)-ionophore-induced secretion in a rat mast cell line

The adenosine analog, N-ethylcarboxamidoadenosine (NECA), causes transient activation of phospholipase C and an enhancement of antigen-induced secretion in a rat mast cell (RBL-2H3) line via adenosine A3-receptors (Ramkumar et al., J. Biol. Chem. 268:16887, 1993) by a mechanism that is inhibited by bacterial toxins and potentiated by dexamethasone (Ali et al., J. Biol. Chem. 265:745-753, 1990). Here we show that NECA synergizes the secretory response to Ca(2+)-ionophore as well as to antigen. The ability of NECA to synergize the secretory responses persisted for 10 to 20 min, long after the early phospholipase C-mediated reactions to NECA had subsided. NECA caused, however, a dose-dependent sustained activation of phospholipase D, as indicated by the formation of [3H]phosphatidic acid, or in the presence of 0.3% ethanol, [3H]phosphatidylethanol. This activation was associated with a sustained increase in diglycerides, in protein kinase C activity and in the phosphorylation of myosin light chains by protein kinase C. The generation of diglycerides was enhanced in dexamethasone-treated cells and suppressed in cells that had been treated with cholera toxin or pertussis toxin. Collectively, the studies suggested that the generation of diglycerides via phospholipase D and the associated activation of protein kinase C were, by themselves, insufficient signals for secretion in RBL-2H3 cells, but that these reactions synergized responses to stimulants such as antigen or A23187 that caused substantial increases in [Ca2+]i.     

A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein

Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.     

Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen

Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.     

A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodeling

Protein kinase C (PKC) plays a major role in regulating cell growth, transformation, and gene expression; however, identifying phosphorylation events that mediate these responses has been difficult. We expression-cloned a group of PKC-binding proteins and identified a high molecular weight, heat-soluble protein as the major PKC-binding protein in REF52 fibroblasts (Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C., and Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422). In this study, we demonstrate that this PKC-binding protein, clone 72, is also a PKC substrate in vitro and in vivo. Using a combination of phosphopeptide mapping, Edman degradation, and electrospray mass spectrometry, serine residues 283, 300, 507, and 515 were identified as the major in vitro PKC phosphorylation sites in clone 72. Phosphorylation state-selective antibodies were raised against phosphopeptides encompassing each of the four phosphorylation sites. These antibodies were used to determine that phorbol esters stimulate phosphorylation of serines 283, 300, 507, and 515 in cultured cells, indicating that clone 72 is directly phosphorylated by PKC in living cells. Phosphorylated clone 72 preferentially accumulates in membrane protrusions and ruffles, indicating that PKC activation and clone 72 phosphorylation are involved in membrane-cytoskeleton remodeling. These data lend further evidence to the model that PKCs directly interact with, phosphorylate, and modify the functions of a group of substrate proteins, STICKs (substrates that interact with C-kinase).     

Dimerization/docking domain of the type Ialpha regulatory subunit of cAMP-dependent protein kinase. Requirements for dimerization and docking are distinct but overlapping

Based on increasing evidence that the type I R subunits as well as the type II R subunits localize to specific subcellular sites, we have carried out an extensive characterization of the stable dimerization domain at the N terminus of RIalpha. Deletion mutants as well as alanine scanning mutagenesis were used to delineate critical regions as well as particular amino acids that are required for homodimerization. A set of nested deletion mutants defined a minimum core required for dimerization. Two single site mutations on the C37H template, RIalpha(F47A) and RIalpha(F52A), were sufficient to abolish dimerization. In addition to serving as a dimerization motif, this domain also serves as a docking surface for binding to dual specificity anchoring proteins (D-AKAPs) (Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) J. Biol. Chem. 272, 8057-8064; Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11184-11189). A similar strategy was used to map the sequence requirements for anchoring of RIalpha to D-AKAP1. Although dimerization appears to be essential for anchoring to D-AKAP1, anchoring can also be abolished by the following single site mutations: C37H, V20A, and I25A. These sites define "hot spots" for the anchoring surface since each of these dimeric proteins are deficient in binding to D-AKAP1. In contrast to earlier predictions, the alignment of the dimerization/docking domains of RIalpha and RII show striking similarities yet subtle differences not only in their secondary structure (Newlon, M. G., Roy, M., Hausken, Z. E., Scott, J. D., and Jennings. P. A. (1997) J. Biol. Chem. 272, 23637-23644) but also in the distribution of residues important for both docking and dimerization functions.     

Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells

We recently demonstrated that transforming growth factor-beta (TGF-beta) stimulates phosphorylation of the type I inositol 1,4, 5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 microM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta1 treatment. TGF-beta1 was also found to stimulate translocation of the alpha-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 microM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.     

A second member of the novel Ca(2+)-dependent protein kinase family from Paramecium tetraurelia. Purification and characterization

The ciliated protozoan Paramecium tetraurelia contains two protein kinase activities (CaPK-1 and CaPK-2) that are dependent on Ca2+ (Gundersen, R. E., and Nelson, D. L. (1987) J. Biol. Chem. 262, 4602-4609). We purified Ca(2+)-dependent protein kinase-1 (CaPK-1) 1,000-fold from the EGTA-extracted soluble fractions of Paramecium. The purified enzyme was a single polypeptide of 52 kDa on SDS-polyacrylamide gel electrophoresis with a native molecular mass of 60,000, suggesting that the active enzyme is a monomer. The purified kinase used casein as the best substrate in vitro, and its activity was absolutely dependent on Ca2+. The physical, catalytic and regulatory properties were clearly distinct from those of casein kinase, protein kinase C, and Ca2+/calmodulin-dependent protein kinases. CaPK-1 was half maximally activated by submicromolar (0.2 microM) free Ca2+, and the purified kinase bound Ca2+ in a blot overlay assay. CaPK-1 and the previously characterized CaPK-2 were biochemically and immunologically different enzymes sharing a similar activation mechanism. CaPK-1 and CaPK-2 appear to be members of a new family of Ca(2+)-dependent protein kinases. A protein immunologically related to the CaPKs was also detected in rat brain.     

Sleep disorders in several pathologic states--endocrine diseases]

Mitochondrial telomere-binding protein (mtTBP) of Candida parapsilosis binds with high affinity to 5' single-stranded overhang of the linear mitochondrial DNA of this yeast (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3056). Here it is reported that mtTBP is phosphorylated by catalytic subunit of cAMP-dependent protein kinase in vitro. Phosphorylated mtTBP has dramatically reduced ability to bind telomeric oligonucleotide in the gel-mobility retardation assay without affecting the oligomerization of mtTBP in vitro. MtTBP is one of the few mitochondrial proteins and the first mitochondrial single-strand DNA binding proteins that was demonstrated to serve as a substrate for cAMP-dependent protein kinase.