Fibrillar amyloid beta-protein forms a membrane-like hydrophobic domain.

Microviscosity of the biological membranes is determined by measuring the fluorescence polarization of diphenylhexatriene (DPH). DPH, a hydrophobic probe, has negligible fluorescence in the solution. When DPH is incorporated into the membrane, it is localized in the membrane hydrophobic core and fluoresces strongly. We report here that DPH also fluoresces in the presence of fibrillar Abeta (fAbeta). However, it does not fluoresce when it is added to the soluble Abeta (sAbeta). DPH inserts into Abeta fibrils in a time-dependent manner, and upon centrifugation, it is sedimented along with fibrils. The steady state fluorescence polarization of DPH with fAbeta1-40 and fAbeta 1-42 was 0.4592 and 0.4898 respectively. These results suggest that fAbeta (but not sAbeta) forms a hydrophobic domain similar to that of membrane.

Interaction of amyloid beta-protein with anionic phospholipids: possible involvement of Lys28 and C-terminus aliphatic amino acids.

Fibrillar amyloid beta-protein (Abeta) is the major protein of amyloid plaques in the brains of patients with Alzheimer's disease (AD). The mechanism by which normally produced soluble Abeta gets fibrillized in AD is not clear. We studied the effect of neutral, zwitterionic, and anionic lipids on the fibrillization of Abeta 1-40. We report here that acidic phospholipids such as phosphatidic acid, phosphatidylserine, phosphatidylinositol (PI), PI 4-phosphate, PI 4,5-P2 and cardiolipin can increase the fibrillization of Abeta, while the neutral lipids (diacylglycerol, cholesterol, cerebrosides), zwitterionic lipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) and anionic lipids lacking phosphate groups (sulfatides, gangliosides) do not affect Abeta fibrillization. Abeta was found to increase the fluorescence of 1-acyl-2-[12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-glycero-3-phosphate (NBD-PA) in a concentration-dependent manner, while no change was observed with 1-acyl-2- [12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl) amino] dodecanoyl]-sn-glycero-3-phosphoethanolamine (NBD-PE). Under similar conditions, other proteins such as apolipoprotein E, gelsolin and polyglutamic acid did not interact with NBD-PA. The order of interaction of amyloid beta-peptides with NBD-PA was Abeta 1-43 = Abeta 1-42 = Abeta 17-42 > Abeta 1-40 = Abeta 17-40. Other Abeta peptides such as Abeta 1-11, Abeta 1-16, Abeta 1-28, Abeta 1-38, Abeta 12-28, Abeta 22-35, Abeta 25-35, and Abeta 31-35 did not increase the NBD-PA fluorescence. These results suggest that phosphate groups, fatty acids, and aliphatic amino acids at the C-terminus end of Abeta 1-40/Abeta 1-42 are essential for the interaction of Abeta with anionic phospholipids, while hydrophilic Abeta segment from 1-16 amino acids does not participate in this interaction. Since positively charged amino acids in Abeta are necessary for the interaction with negatively charged phosphate groups of phospholipids, it is suggested that Lys28 of Abeta may provide anchor for the phosphate groups of lipids, while aliphatic amino acids (Val-Val-Ile-Ala) at the C-terminus of Abeta interact with fatty acids of phospholipids.

Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils.

Amyloid beta-protein (Abeta) is present in soluble form in the plasma and cerebrospinal fluid (CSF) of normal people and patients with Alzheimer's disease (AD). However, in AD patients, Abeta gets fibrillized as the main constituent of amyloid plaques in the brain. Soluble synthetic Abeta also forms amyloid-like fibrils when it is allowed to age. The mechanism that prevents soluble Abeta from fibrillization in biological fluids is not clear. We recently reported that gelsolin, a secretory protein, binds to Abeta, and that gelsolin/Abeta complex is present in the plasma [V.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Biochem. Biophys. Res. Commun. 258 (1999) 241-246.]. We now studied the effect of gelsolin on Abeta fibrillization. Congo red staining and electron microscopic examination in negative staining of aged samples of Abeta alone and Abeta incubated with gelsolin showed that gelsolin inhibits the fibrillization of synthetic Abeta 1-40 and Abeta 1-42 at gelsolin to Abeta molar ratio of 1:40. In addition, gelsolin also defibrillized the preformed fibrils of Abeta 1-40 and Abeta 1-42 in a time-dependent manner. These results suggest that gelsolin functions as an anti-amyloidogenic protein in the plasma and CSF, where it prevents Abeta from fibrillization, and helps to maintain it in the soluble form.

Binding of gelsolin, a secretory protein, to amyloid beta-protein.

Soluble amyloid beta-protein (Abeta) is normally present in the cerebrospinal fluid (CSF) and plasma. However, it is fibrillized and deposited as plaques in the brains of patients with Alzheimer's disease. Cerebrospinal fluid (CSF) contains several circulating proteins (apolipoprotein E, apolipoprotein J, and transthyretin) that bind to Abeta. We report here that gelsolin, a secretory protein, also binds to Abeta in a concentration-dependent manner. Under similar conditions, other proteins such as G-actin, protein kinase C, polyglutamic acid, and gelatin did not bind to Abeta. Solid phase binding assays showed two Abeta binding sites on gelsolin that have dissociation constants (Kd) of 1.38 and 2.55 microM. Abeta was found to co-immunoprecipitate along with gelsolin from the plasma, suggesting that gelsolin-Abeta complex exists under physiological conditions. The gelsolin-Abeta complex was sodium dodecyl sulfate (SDS)stable in the absence of reducing agent, but was dissociated when the SDS stop solution contained dithiothreitol (reducing agent). This study suggests that the function of secretory gelsolin in the CSF and plasma is to bind and sequester Abeta.

Binding of amyloid beta-protein to intracellular brain proteins in rat and human.

Amyloid beta-protein (Abeta), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of Abeta to intracellular proteins has not been studied. We have developed an overlay assay to study Abeta binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to Abeta 1-40 and Abeta 1-42. No major difference was observed in the Abeta binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of Abeta to brain proteins resides between 12-28 amino acid sequence of Abeta. The presence of several intracellular Abeta-binding (AbetaB) proteins suggests that these proteins may either protect Abeta from its fibrillization or alternatively promote Abeta polymerization. Identification of these proteins and their binding affinities for Abeta are needed to assess their potential role in the pathogenesis of Alzheimer's disease.

Aggregation of amyloid beta-protein as function of age and apolipoprotein E in normal and Alzheimer's serum.

We compared the effect of serum from (a) 26 Alzheimer's disease (AD) patients and 22 age-matched non-demented controls (CO) with apolipoprotein E 4/4, 3/3 or 3/2 phenotypes, and (b) 17 normal young (aged 15-41 years) and 21 normal elderly (aged 64-83 years) people on in vitro aggregation of synthetic amyloid beta-protein (A beta) 1-40 by Thioflavin T fluorescence spectroscopy. A beta 1-40 aggregation in presence of serum from the normal elderly group was significantly higher as compared to the normal young group (correlation coefficient between age and A beta aggregation=0.73). However, no difference in A beta aggregation was observed in the presence of serum from AD patients and non-demented controls. There was a positive correlation between serum apo E concentrations and A beta aggregation, while there was no significant difference between different apo E phenotypes. The correlation coefficient in the AD 4/4 (0.65) was higher than the CO 4/4 group (0.04), while it was lower in the AD 3/3 group (-0.12) than in the CO 3/3 (0.39) group. These results suggest that the apo E4 allele alone may not be responsible for A beta fibril formation in AD; other factors may be involved in increasing risk for AD pathogenesis in those having the apo E4 allele. The severity of dementia and serum albumin levels also did not correlate with A beta aggregation. We propose that the age of an individual may be an important factor in determining the degree of A beta aggregation/fibrillization, and that mechanism of sequestration of A beta in serum may not be defective in AD.

Two nonmuscle myosin II heavy chain isoforms expressed in rabbit brains: filament forming properties, the effects of phosphorylation by protein kinase C and casein kinase II, and location of the phosphorylation sites.

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIB alpha and MIIB beta, in rabbit brains. The B alpha form corresponded to SMemb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B beta form was novel. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms. Casein kinase II (CK II) incorporated 4 and 2 mol of phosphate/mol of MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites in MIIB beta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4)-, with total incorporation of about 2 mol/mol of peptide. In addition, PK C phosphorylated a Ser within the predicted helical domain, E-V-S(PO4)-T-L, in both MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4) -E. The same sites were phosphorylated in MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) the helical domains dictate the intrinsic salt dependence of assembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH-terminal nonhelical domain, and (3) the effects of the phosphorylation on assembly are isoform specific.

Metal cations defibrillize the amyloid beta-protein fibrils.

Amyloid beta-protein (A beta) is the major constituent of amyloid fibrils composing beta-amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A beta by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A beta 1-40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A beta 1-40 fibrils by metal cations was: Ca2+ and Zn2+ (IC50 = 100 microM) > Mg3+ (IC50 = 300 microM) > Al3+ (IC50 = 1.1 mM). EM analysis in negative staining showed that A beta 1-40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca2+, Mg2+, and Zn2+ to preformed A beta 1-40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al3+ was less effective, and reduced the fibril network by about 80% of that in the absence of any metal cation. Studies with A beta 1-42 showed that this peptide forms more dense network of fibrils as compared to A beta 1-40. Both ThT fluorescence spectroscopy and EM showed that similar to A beta 1-40, A beta 1-42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca2+. These studies suggest that metal cations can defibrillize the fibrils of synthetic A beta.

Media from rhabdomyosarcoma and neuroblastoma cell cultures stimulate in vitro aggregation and fibrillization of amyloid beta-protein.

In vitro aggregation and fibrillization of synthetic amyloid beta-protein Abeta 1-40 was assessed in the conditioned media from rhabdomyosarcoma (CRL 1598, HTB 82, HTB 153, CCL 136), adenocarcinoma (CCL 218), neuroblastoma (SY5Y), and COS cells cultured in the absence and presence of 10% heat-inactivated fetal bovine serum (FBS). The aggregation and formation of cross beta-pleated sheet structures in Abeta was quantitated by Thioflavin T (ThT) fluorescence spectroscopy, while the morphology of Abeta fibrils was examined in negative staining in the electronmicroscope (EM). In cultures supplemented with 10% FBS, the conditioned media from CRL 1598, HTB 82, CCL 218, and SY5Y cell cultures stimulated Abeta aggregation in a time-dependent manner as compared to that of control (serum-containing medium that had not been exposed to cells). The order of stimulation was SY5Y > CRL 1598 > or = HTB 82 > CCL 218, and the stimulation was higher in 2 week cultures than in 1 week cultures. Similar studies using media from HTB 153, CCL 136 and COS cell cultures showed no effect on Abeta 1-40 aggregation. In serum-free cell cultures, only media from SY5Y and CRL 1598 could promote significant aggregation of Abeta 1-40. Negative staining in EM revealed Abeta fibril formation only with conditioned media from SY5Y and CRL 1598 cultured under serum free conditions; no Abeta fibrils were noticed in media from cell cultures supplemented with 10% FBS. We propose that both the SY5Y neuroblastoma cell line and the CRL 1598 rhabdomyosarcoma cell line may serve as experimental models for in vitro studies of extracellular aggregation and fibrillization of Abeta-protein in cell cultures, while rhabdomyosarcoma HTB 82 and adenocarcinoma CCL 218 may be models for study of Abeta aggregation only.

Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity.

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also has protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase was not affected in the presence of profilin. Kinetic analysis, as a function of varying concentrations of ATP and PI 4,5-P2, showed that profilin affects the Vmax of PI 3-kinase without affecting k(m). Profilin may also affect PI 3-kinase activity by its direct association to the enzyme because dot-blot analysis using antibody to glutathione S-transferase (GST) suggested that GST-85 kDa, a fusion protein of PI 3-kinase, binds to profilin. However, PI 3-kinase did not affect the actin-sequestering ability of profilin (determined by pyrene-labeled actin), which indicates that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate the actin polymerization.

Effect of cerebrospinal fluid from normal and Alzheimer's patients with different apolipoprotein E phenotypes on in vitro aggregation of amyloid beta-protein.

We examined the effect of cerebrospinal fluid (CSF) from 23 Alzheimer's disease (AD) patients and 22 age-matched non-demented controls with apolipoprotein E4/4, 3/3, or 3/2 phenotypes on in vitro aggregation of amyloid beta-protein (A beta) 1-40 by Thioflavin T fluorescence spectroscopy. CSF from both AD and control groups inhibited A beta aggregation, as compared to that of phosphate buffered saline, in agreement with an earlier report (Wisniewski et al., 1993). However, there was significantly less aggregation of A beta in presence of CSF from AD than that from non-demented controls. The presence of CSF from controls with apoE3/3 phenotype resulted in higher A beta aggregation as compared to other phenotypes. There was a positive correlation between CSF apoE concentrations and A beta aggregation; whereas age, CSF soluble A beta levels or severity of dementia did not correlate with A beta aggregation. These results suggest that mechanism of sequestration of A beta in CSF may not be defective in AD. Amyloid formation in AD may be impact of altered balance of other factors such as amyloid-associated proteins/extracellular matrix components that can immobilize A beta in the brain, and promote its fibrillogenesis in AD.

Phosphoinositide-dependent in vitro phosphorylation of profilin by protein kinase C. Phospholipid specificity and localization of the phosphorylation site.

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).

Phospholipid binding, phosphorylation by protein kinase C, and filament assembly of the COOH terminal heavy chain fragments of nonmuscle myosin II isoforms MIIA and MIIB.

Previously, we showed that myosin II heavy chains bind to phosphatidylserine (PS) liposomes via their COOH terminal regions and that protein kinase C (PK C) phosphorylates the PS-bound heavy chains [Murakami et al. (1994) J. Biol. Chem. 269, 16082-16090]. In this report, we studied the phospholipid binding, the kinetics of phosphorylation by PK C, and the effect of PK C-mediated phosphorylation on assembly using 46-47 kDa fragments from the COOH termini of macrophage (MIIAF46) and brain type (MIIBF47) heavy chain isoforms. Binding of the fragments to PS or phosphatidylinositol liposomes increased turbidity, but MIIAF46 gave higher turbidity than MIIBF47. Both fragments were sedimented similarly by ultracentrifugation in PS concentration and mole percent of PS dependent manners. With mixed PS/phosphatidylcholine (PC) liposomes, at least 70 mol % PS was required for heavy chain binding. A similar level of PS was required for phosphorylation of fragments by PK C, indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PK C. PK C phosphorylated MIIBF47 with Vmax values 4-5 times higher than those of MIIAF46, but the Km values for the two substrates were similar. The apparent Km values for PS liposomes (Klipid) were also similar for phosphorylation of both isoforms. Mixing PS with PC increased the Klipid and reduced the Vmax values but did not alter the Km values for the substrates. Assembly of MIIBF47, but not MIIAF46, was significantly inhibited by the phosphorylation, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PK C.

Phosphatidylinositol 3-kinase: inhibition of intrinsic protein-serine kinase activity by phosphoinositides, and of lipid kinase activity by Mn2+.

Phosphatidylinositol (PI) 3-kinase is composed of 110 kDa catalytic and 85 kDa regulatory subunits. The 110 kDa subunit has two intrinsic kinase activities, i.e., Mn(2+)-dependent protein-serine kinase and Mg(2+)-dependent lipid kinase activities. These intrinsic kinases have been reported to be interdependent: protein-serine kinase phosphorylates the 85 kDa subunit of PI 3-kinase, which upon phosphorylation inhibits the lipid kinase activity of PI 3-kinase. We report here that phosphoinositides can selectively inhibit the protein-serine kinase activity of PI 3-kinase without affecting lipid kinase activity. This inhibition depends on the phosphorylation status of the phosphoinositides, i.e., PI 4,5-bisphosphate > PI 4-phosphate >> PI. Mn2+ (2 mM) protected protein kinase activity from phosphoinositides-mediated inhibition if added prior to interaction of PI 3-kinase with phosphoinositides. On the other hand, Mn2+ (2 mM) inhibited lipid kinase activity independent of its effect on the protein kinase activity of PI 3-kinase. The present study suggests that the protein-serine kinase and the lipid kinase activities of PI 3-kinase can be selectively inhibited by phosphoinositides and Mn2+ respectively.

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase.

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occurring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC50 of 100 microM. On the other hand, spermine (5 mM) stimulated the activity of PIP 5-kinase 2-3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg2+ and PIP2 concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP2 concentrations. While 1 mM calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg2+ (1.25 mM), inhibition was observed at optimal concentration of Mg2+ (2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.

Direct binding of myosin II to phospholipid vesicles via tail regions and phosphorylation of the heavy chains by protein kinase C.

Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation site within their tail regions. A fragment of human macrophage myosin heavy chain, encompassing its COOH-terminal 396 amino acids (MIIAF46), was expressed in Escherichia coli to provide a model system for study of PKC-mediated phosphorylation. PKC phosphorylated this fragment when phosphatidylserine (PS) liposomes were present, but not when liposomes made from PS/phosphatidylcholine (PC) were used. The reaction required Ca2+, but not other activators such as diacylglycerol (DG) or phosphatidylinositol 4,5-bisphosphate. Phosphorylation of MIIAF46 was not observed in the presence of micelles of PS or PS/DG. Similar results were obtained using native myosin II purified from bovine brain and chicken intestine brush border. Phosphorylation of light chains, in contrast, occurred even with PS/PC liposomes if DG was present. Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIAF46 and native myosin II, and the extent of increase depended upon the type of myosin used. Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIAF46 and myosin II. In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight increase in turbidity with all myosins and fragments and did not noticeably shift their gel filtration elution positions. These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, that the binding is dependent upon the PS composition, and that PKC phosphorylates the PS-bound heavy chains.

Interaction of protein kinase C and phosphoinositides: regulation by polyamines.

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates protein kinase C (PKC) in the presence of phosphatidylserine and calcium. Recently it has been demonstrated that direct interaction of PKC with PIP2 in the absence of divalent cation inactivates this kinase. In the present study, the interaction of natural aliphatic polyamines with phosphoinositides was investigated for its possible relevance to PKC-mediated protein phosphorylation. PKC/phosphoinositide interaction was studied by monitoring the changes in (a) intrinsic fluorescence of the enzyme, and (b) PKC activity (protamine sulphate or histone III-S as substrate). All the phosphoinositides: PIP2, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol (PI) inactivated PKC with an IC50 of 0.4 microM for PIP2, 5 microM for PIP and 10 microM for PI. Hydrogenated PIP2 behaved similarly to that of natural PIP2. Time-dependent studies showed very rapid inactivation of PKC by PIP2. The polyamines spermine and spermidine at physiological concentrations protected PKC from phosphoinositides-mediated inactivation when added prior to PKC interaction with phosphoinositides. Putrescine was least effective. Addition of spermine or spermidine to PKC/phosphoinositides incubation mixture did not reverse PKC activity indicating that the inactivation of PKC by phosphoinositides is irreversible. Fluorescence quenching experiments showed that phosphoinositides inactivate PKC by inducing conformational changes of the enzyme that are prevented by spermine. We propose that polyamines protect PKC and possibly other protein kinase from phosphoinositides-mediated inactivation, and that inactivation of protein kinases by phosphoinositides may not have physiological relevance.

Amyloid beta-protein stimulates casein kinase I and casein kinase II activities.

Amyloid beta-protein (A beta) is the major protein of cerebrovascular and plaque amyloid in Alzheimer's disease (AD). Extensive evidence has demonstrated abnormal protein phosphorylation in this disease. We investigated the effect of synthetic A beta with the amino-acid sequence corresponding to cerebrovascular A beta and plaque A beta on the activities of casein kinase I (CK I) and casein kinase II (CK II). These enzymes were purified from bovine brain and casein was used as a substrate. A beta was found to stimulate markedly CK I- and CK II-mediated phosphorylation of casein in a concentration-dependent manner. The effect of plaque A beta was considerably higher than that of cerebrovascular A beta. Heparin, which is known to be a specific inhibitor of CK II, completely inhibited A beta-stimulated CK II activity. A beta itself was not a substrate for casein kinases. These findings were confirmed using other substrates for CK I and CK II. The experiments with synthetic CK II-substrate peptide (Leu-Glu-Leu-Ser-Asp-Asp-Asp-Asp-Glu) and the phosphorylation of erythrocyte membrane proteins by intrinsic membrane-bound CK I in erythrocytes showed marked stimulation in activities of casein kinases in the presence of A beta 1-40 or blocked A beta. We propose that A beta, by stimulating casein kinases, may contribute to abnormal protein phosphorylation in AD, in particular to increased phosphorylation of microtubule-associated proteins, leading to the neurofibrillary tangles formation and neurodegeneration in this disease. Interaction of A beta with protein kinases, thus, may characterize the beginning of the disease.

Activation of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.

Phosphatidylinositol 3-kinase (PI 3-kinase) was partially purified from rat liver cytosol and used to synthesize phosphatidylinositol 3,4,5-trisphosphate (PIP3), using phosphatidylinositol 4,5-bisphosphate (PIP2) as a substrate. Purified PIP3 (free of chromatographic oxalate) activated protein kinase C (PKC) in the presence of phosphatidylserine and calcium (PKC -cofactors) in a concentration-dependent manner. In the absence of these cofactors, effect of PIP3 was not observed. Comparison of the effects of PIP3 and PIP2 on PKC activity indicates that PIP3 is a more potent PKC-activator than PIP2. The affinity of PKC to PIP3 was 4 fold higher than that to PIP2 (KPIP3 = 0.022 and KPIP2 = 0.087 mol %), while its maximal velocity (Vmax) was similar to that of PIP2-stimulated PKC activity (0.4 - 0.5 mumol/mg/min). These results suggest a physiological role for PIP3 in signal transduction, and support the previous finding (Chauhan et al. (1991) Arch. Biochem. Biophys. 287,283) that PKC-activation by phosphoinositides increases with the state of phosphorylation of these lipids. We propose that PIP3 by activating PKC may initiate a cascade of events from PIP3-->PKC- activation-->effects on other protein kinases such as MAP-kinase-->gene expression.