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.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.

Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.     

Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain.

Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with neuromodulin and also corresponds to the PKC phosphorylation site on neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes neuromodulin. We propose that the amino acid sequence shared by neurogranin and neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.     

Regulation of endothelial nitric oxide synthase by protein kinase C

Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.     

Protein kinase C substrates from bovine brain. Purification and characterization of neuromodulin, a neuron-specific calmodulin-binding protein

Although such solubility is uncommon among proteins generally, several bovine brain proteins were found to be soluble in 2.5% perchloric acid, and many of them were in vitro substrates for protein kinase C (Ca2+/phospholipid-dependent enzyme). Two of the perchloric acid-soluble brain proteins were purified, p43 and p17. P43 and p17 could be phosphorylated by protein kinase C only in the presence of Ca2+ and phospholipids and neither was a substrate for protein kinase II. P43 was subsequently identified as the neurospecific, calmodulin-binding protein, neuromodulin (also designated P-57, GAP43, B50, or F1) (Alexander, K. H., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544-7549). A rapid purification method for neuromodulin was developed taking advantage of its newly discovered property, solubility in 2.5% perchloric acid, and of its previously recognized calmodulin-binding property. Evidence was obtained that neuromodulin isolated from cytosolic extract exists as a mixture of molecular forms and that the Ca2+-binding S100 protein-beta discriminates among the different neuromodulin isoforms in forming covalent complexes via disulfide bridges; this discrimination may be explained by analogous differences observed between the NH2-terminal amino acid sequences of p57 and F1. Solubility in 2.5% perchloric acid was demonstrated for another rat brain protein kinase C substrate, p87. We suggest that perchloric acid solubility might be a common property of protein kinase C substrates.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

Identification of two domains which mediate the binding of activating phospholipids to the plasma-membrane Ca2+ pump.

The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid-responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070-8076] and to the calmodulin-binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin-binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca(2+)-ATPase lacking the calmodulin-binding domain confirmed that the phospholipid-induced stimulation is mediated by two sites, one located in the C-terminal portion of the previously identified 44-amino-acid phospholipid-responsive domain, the other in the calmodulin-binding domain.     

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.     

Dephosphorylation of neuromodulin by calcineurin

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.     

Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate.

Protein kinases and phosphatases are targeted through association with anchoring proteins that tether the enzymes to subcellular structures and organelles. Through in situ fluorescent techniques using a Green Fluorescent Protein tag, we have mapped membrane-targeting domains on AKAP79, a multivalent anchoring protein that binds the cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and protein phosphatase 2B, calcineurin (CaN). Three linear sequences termed region A (residues 31-52), region B (residues 76-101) and region C (residues 116-145) mediate targeting of AKAP79 in HEK-293 cells and cortical neurons. Analysis of these targeting sequences suggests that they contain putative phosphorylation sites for PKA and PKC and are rich in basic and hydrophobic amino acids similar to a class of membrane-targeting domains which bind acidic phospholipids and calmodulin. Accordingly, the AKAP79 basic regions mediate binding to membrane vesicles containing acidic phospholipids including phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P2] and this binding is regulated by phosphorylation and calcium-calmodulin. Finally, AKAP79 was shown to be phosphorylated in HEK-293 cells following stimulation of PKA and PKC, and activation of PKC or calmodulin was shown to release AKAP79 from membrane particulate fractions. These findings suggest that AKAP79 might function in cells not only as an anchoring protein but also as a substrate and effector for the anchored kinases and phosphatases.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.     

Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.     

Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.