Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.     

Cross-reactivity of T-cell clones specific for altered peptide ligands of myelin basic protein

We have determined that certain altered peptide ligands (APLs) can induce T-cells specific for the native peptide myelin basic protein (MBP) p85-99 to secrete Th2-type cytokines such as IL-4 and IL-5 in the absence of significant Th1-type cytokines. However, it is not known whether stimulation with APLs will activate autoreactive T cells or a distinct population of cells. In the present study, 18 T-cell clones that reacted with either MBP p85-99 or one of three APLs of the peptide substituted at TCR contact residues were generated. T-cells were tested functionally for their reactivity to the original stimulating peptide as well as to the MBP APLs. In addition, the T-cell receptor (TCR) alpha and beta chains of each of these clones were sequenced. In a series of T-cell clones isolated from a multiple sclerosis patient, stimulation of T-cells with the APL 93A, which has an alanine for lysine substitution at the TCR contact residue 93, did not induce substantial proliferation of MBPp85-99-specific T-cell clones, indicating that a distinct set of T-cell clones was induced. However, this was not the case for another set of T-cell clones from a different individual in which the 93A peptide induced clonal expansion of T-cells highly reactive with the native MBPp85-99 antigen. Thus, the potential beneficial effect of using APLs to induce downregulatory cytokines appears to depend on the specific T-cell repertoire of the individual patient.     

Substrate-dependent inhibition of protein kinase C by specific inhibitors

Protein kinase C (PKC) and its proteolysis-derived protein kinase independent of Ca2+ and phospholipids (PKM), were purified from rat brain. By using histone H1 and protamine as substrates, we assayed the effect of several inhibitors of PKC and PKM. The inhibition turned out to be dependent on both the nature of the kinase and the type of substrate assayed. These results may help to interpret the different responses elicited by PKC inhibitors in vivo.     

Phosphorylation of coagulation factor II by phospholipid/Ca(2+)-dependent protein kinase (protein kinase C)

Prothrombin is a major constituent of the blood coagulation cascade and requires phospholipid and Ca2+ for its activation. We have found that phospholipid/Ca(2+)-dependent protein kinase (Protein kinase C) phosphorylates prothrombin and the associated apparent Km value for prothrombin (0.86 microM) is comparable to the Km value reported for most known substrates of protein kinase C. A 2-dimension separation analysis revealed that serine residue was apparently phosphorylated by PKC. The phosphorylation was inhibited by such phosphatidylserine- and/or Ca2+ competitive protein kinase C inhibitors as trifluoperazine, palmitoylcarnitine and gossypol. These results suggest that protein kinase C phosphorylation was involved in the regulation of blood coagulation.     

Rice small C2-domain proteins are phosphorylated by calcium-dependent protein kinase

We previously reported that OsERG1 and OsERG3 encode rice small C2-domain proteins with different biochemical properties in Ca²⁺- and phospholipid-binding assays. Os-ERG1 exhibited Ca²⁺-dependent phospholipid binding, which was not observed with OsERG3. In the present study, we show that both OsERG1 and OsERG3 proteins exhibit oligomerization properties as determined by native polyacrylamide gel electrophoresis (PAGE) and glutaraldehyde cross-linking experiments. Furthermore, in vitro phosphorylation assays reveal the phosphorylation of OsERG1 and OsERG3 by a rice calcium-dependent protein kinase, OsCDPK5. Our mutation analysis on putative serine phosphorylation sites shows that the first serine (Ser) at position 41 of OsERG1 may be an essential residue for phosphorylation by OsCDPK5. Mutation of Ser41 to alanine (OsERG1S41A) and aspartate (OsERG1S41D) abolishes the ability of OsERG1 to bind phospholipids regardless of the presence or absence of Ca²⁺ ions. In addition, unlike the OsERG1 wild-type form, the mutant OsERG1 (S41A)::smGFP construct lost the ability to translocate from the cytosol to the plasma membrane in response to calcium ions or fungal elicitor. These results indicate that Ser41 may be essential for the function of OsERG1.     

Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C

The phosphorylation of synthetic peptides derived from the NH₂-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1–13) (MLC(1–13)). Kinetic analysis of MLC(1–13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1–13) had a V/K of 2.4 min⁻¹·mg⁻¹, whereas the V/K of MLC(3–13) was 3.0 min⁻¹·mg⁻¹. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent V_max. Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent K_m increased 7-fold and the V_max decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase

The substrate specificity of protein kinase C was studied and compared with that of cyclic AMP-dependent protein kinase (protein kinase A) by using bovine brain myelin basic protein as a model substrate. This basic protein was phosphorylated at multiple sites by both of these protein kinases. In this analysis, the basic protein was thoroughly phosphorylated in vitro with [gamma-32P]ATP and each protein kinase, and then digested with trypsin. The resulting radioactive phosphopeptides were isolated by gel filtration followed by high performance liquid chromatography on a reverse-phase column. Subsequent amino acid analysis and/or sequential Edman degradation of the purified phosphopeptides, together with the known primary sequence of this protein, revealed that Ser-46 and Ser-151 were specifically phosphorylated by protein kinase C, whereas Thr-34 and Ser-115 were phosphorylated preferentially by protein kinase A. Both kinases reacted with Ser-8, Ser-11, Ser-55, Ser-110, Ser-132, and Ser-161 at various reaction velocities. Contrary to protein kinase A, protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine. The seryl residues that are phosphorylated commonly by these two protein kinases have basic amino acids at both the amino- and carboxyl-terminal sides. These results provide some clues to understanding the rationale that these kinases may show different but sometimes similar functions depending on the structure of target phosphate acceptor proteins.     

Protein kinase C-dependent activation of a myelin basic protein kinase by gastrin-releasing peptide in Swiss 3T3 fibroblasts

Addition of gastrin releasing peptide to serum-starved Swiss 3T3 mouse fibroblasts results in a transient appearance of a myelin basic protein-kinase activity in cytosolic extracts. Increased kinase activity is also observed upon stimulation of cells with bradykinin, epidermal growth factor or 4 beta-phorbol dibutyrate. Chromatographic analysis of the cytosolic extracts show that both gastrin-releasing peptide and 4 beta-phorbol dibutyrate induce the appearance of a kinase activity similar to that induced by epidermal growth factor. The response to gastrin-releasing peptide is abolished by down-regulation of protein kinase C and attenuated by acute inhibition of protein kinase C using staurosporine. The effect of epidermal growth factor was also suppressed under these conditions, albeit to a lesser extent. The results indicate (1) that activation of myelin basic protein kinase(s) may be common to different growth factors, and (2) that protein kinase C may participate in this response, at least in the case of gastrin-releasing peptide.     

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Inositol phospholipid turnover and protein kinase C translocation are stimulated by poly(I).poly(C) in human amnion cells (UAC)

Polyinosinic-polycytidylic acid, a potent inducer of inducer of interferon (IFN) production and activator of some IFN-induced enzymes, inhibits [3H]uridine incorporation into the RNA of vesicular stomatitis virus even in the absence of IFN synthesis, transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C. Since IFNs also have similar activities these results suggest that IFN induction and IFN function are realised through common biochemical pathways.     

Retinal inhibits TPA activated, calcium-dependent, phospholipid-dependent protein kinase ("C" kinase)

RAW264 macrophage-like cells contain a kinase which is dependent on Ca++ and phosphatidylserine for activity (C kinase) and is stimulable by the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate. Retinal inhibits the activity of the tumor promoter-activated kinase in a concentration-dependent manner. The apparent Ki for inhibition is 1 X 10(-5)M. Retinal is not a general inhibitor of phosphotransferase reactions as it did not inhibit the activity of purified cyclic AMP-dependent protein kinase. It is possible, therefore, that the action of retinoids to antagonize tumor promoter effects on cell function may be mediated at the level of regulation of C-kinase.     

Synthetic segments of the mammalian beta AR are preferentially recognized by cAMP-dependent protein kinase and protein kinase C

Desensitization of the beta-adrenergic receptor has been correlated in some cell systems with receptor phosphorylation. Various kinases have been implicated in these phosphorylation processes, including both cAMP-dependent protein kinase and protein kinase C. In the present study, we have utilized the protein sequence information obtained from the cloning of the mammalian beta-adrenergic receptor to prepare synthetic peptides corresponding to regions of the receptor which would be predicted to act as possible substrates for these kinases in vivo. Two of these receptor-derived peptides were found to serve as substrates for these protein kinases. A peptide corresponding to amino acids 257-264 of the beta-receptor is the preferred substrate for the cAMP-dependent protein kinase, while protein kinase C showed a marked preference for phosphorylation of a peptide corresponding to residues 341-351 of the beta-adrenergic receptor.     

Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase p44mpk.

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein-serine/threonine kinase (p44mpk) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid-phase sequencing by Edman degradation identified the major site as Thr-97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91-104 in bovine brain myelin basic protein. Thr-94 was also phosphorylated by p44mpk to a very minor extent.     

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.     

Association of protein kinase C with phospholipid vesicles

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+ -dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g of phospholipid at steric saturation. Binding was of relatively high affinity (Kd less than 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of dodecylphosphocholine/myelin basic protein complexes

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.