PKCλ is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP

Direct phosphorylation of GluA1 by PKC controls α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA) receptor (AMPAR) incorporation into active synapses during long‐term potentiation (LTP). Numerous signalling molecules that involved in AMPAR incorporation have been identified, but the specific PKC isoform(s) participating in GluA1 phosphorylation and the molecule triggering PKC activation remain largely unknown. Here, we report that the atypical isoform of PKC, PKCλ, is a critical molecule that acts downstream of phosphatidylinositol 3‐kinase (PI3K) and is essential for LTP expression. PKCλ activation is required for both GluA1 phosphorylation and increased surface expression of AMPARs during LTP. Moreover, p62 interacts with both PKCλ and GluA1 during LTP and may serve as a scaffolding protein to place PKCλ in close proximity to facilitate GluA1 phosphorylation by PKCλ. Thus, we conclude that PKCλ is the critical signalling molecule responsible for GluA1‐containing AMPAR phosphorylation and synaptic incorporation at activated synapses during LTP expression.     

Increased Phosphorylation of a 17-kDa Protein Kinase C Substrate (P17) in Long-Term Potentiation

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.     

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts.

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.     

Protected-Site Phosphorylation of Protein Kinase C in Hippocampal Long-Term Potentiation

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca²⁺, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

Ganglioside-modulated protein phosphorylation in myelin

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.     

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.     

Protein kinase C in rat brain myelin

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca²⁺ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.     

The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein

The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence –T92-P93-R94-T95-P96-P97-P98-S99–) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72–S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95.     

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulose cells: Involvement of cGMP-dependent and independent signal transduction mechanisms

To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1×10^–7 M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca²⁺-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca²⁺ (0.5 mM), the incorporation of³²P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca²⁺-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PCK were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca²⁺ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and-independent manner in bovine adrenal glomerulosa cell membranes. (Mol Cell Biochem141: 103–111, 1994)     

Activated protein kinase C directly phosphorylates the CD34 antigen on hematopoietic cells.

The CD34 antigen is a human leukocyte membrane protein expressed specifically by lymphohematopoietic progenitor cells. We found that CD34 is a phosphoprotein and therefore examined the regulation of its phosphorylation. Activation of protein kinase C (PKC) enhanced CD34 phosphorylation. The PKC activators, 12-O-tetradecanoylphorbol-13-acetate and bryostatin-1, induced rapid, stoichiometric hyperphosphorylation of CD34 protein in cells, resulting in a 5-fold increase in CD34 phosphorylation. In vitro kinase studies revealed that purified PKC could directly phosphorylate purified CD34. Only serine phosphorylation was detected in the CD34 molecule. Two-dimensional phosphopeptide mapping experiments indicated that PKC induces the phosphorylation of identical serine residue(s) in vitro and in vivo (in KG1 cells). These are newly phosphorylated serine residue(s), which are not detectably phosphorylated in CD34 from exponentially growing KG1 cells. These data indicate that the developmental stage-specific molecule, CD34, is a phosphorylation target for activated PKC. Furthermore, these findings raise the possibility that PKC activation and phosphorylation of the CD34 molecule may play a role in signal transduction during early lymphohematopoiesis.     

Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase.

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.     

Role of PKCζ‐NADPH oxidase signaling axis in PKCα‐mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.     

Phosphorylation of pleckstrin increases proinflammatory cytokine secretion by mononuclear phagocytes in diabetes mellitus

The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-alpha and IL-1beta) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.     

Myelin management by the 18.5‐kDa and 21.5‐kDa classic myelin basic protein isoforms

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5‐kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two‐dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3‐domains, participation in Fyn‐mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post‐translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5‐kDa MBP's protein‐membrane and protein‐protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5‐kDa MBP has significant roles beyond membrane adhesion. The full‐length, early‐developmental 21.5‐kDa splice isoform is predominantly karyophilic due to a non‐traditional P‐Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.     

The antioxidant butylated hydroxytoluene (BHT) inhibits the dioctanoylglycerol-evoked platelet response but potentiates that elicited by ionomycin.

Preincubation of aspirin-treated human platelets with butylated hydroxytoluene (BHT) inhibits secretion, aggregation, and protein phosphorylation induced by dioctanoylglycerol or phorbol 12-myristate 13-acetate (PMA). BHT alone elicits a rapid and transient phosphorylation of a 47-kDa protein, which is indistinguishable from the well-recognized major substrate of protein kinase C (PKC). Inhibition of diacylglycerol- or PMA-induced platelet activation is also observed after decay to the basal level of the BHT-evoked phosphorylation of the 47-kDa protein. By contrast BHT potentiates platelet responses elicited by the calcium ionophore ionomycin. In the presence of the PKC inhibitor staurosporine BHT fails to increase the ionomycin-promoted platelet aggregation, indicating that its effect occurs through a PKC activation, even if no correlation with the 47-kDa protein phosphorylation is observed. BHT does not significantly modify the affinity of protein kinase C purified from calf brain for Ca2+ or dioctanoylglycerol. It is concluded that: (a) a short exposure of platelets to BHT induces an activation, whereas a long exposure an inhibition of PKC, (b) at variance with diacylglycerols BHT decreases the platelet responses promoted by subsequent challenge with PKC activators themselves, and (c) similarly to other PKC activators BHT potentiates the cellular response elicited by calcium ionophores most likely by activating the phospholipase A2.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.