H. Zwiers

Phosphorylation of B 50 protein by calcium activated, phospholipid dependent protein kinase and B 50 protein kinase

Abstract: B‐50 is a brain‐specific phosphoprotein, the phosphorylation state of which may play a role in the regulation of (poly)phosphoinositide metabolism. Several kinases were tested for their ability to phosphorylate purified B‐50 protein. Only calcium‐activated, phospholipid‐dependent protein kinase (kinase C) and B‐50 protein kinase were able to use B‐50 protein as a substrate. Furthermore, kinase C specifically phosphorylates B‐50 when added to synaptic plasma membranes. We further characterized the sensitivity of kinase C and B‐50 kinase to ACTH (and various fragments), phospholipids, chlorpromazine, and proteolytic activation. Since the sensitivities of both kinases were similar, we conclude that B‐50 protein kinase is a calcium‐dependent, phospholipidstimulated protein kinase of the same type as kinase C.

Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism

This study describes the ultrastructural localization in rat hippocampal tissue in situ and in isolated synaptosomes of the brain-specific phosphoprotein B-50, using affinity purified anti-B-50 immunoglobulins (IgGs). Evidence is presented for the presynaptic localization of B-50 in rat brain. Given this specific localization a model is presented outlining the presumed function of the B-50 protein in the membrane and describing possible neuromodulation by adrenocorticotropin hormone (ACTH)-like peptides.

ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated32P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10−10 to 10−4 M. In a number of protein bands, a biphasic effect of ACTH1–24 was observed: in concentrations of 10−4–10−5 M, a reduced amount of32P was found; in concentrations of 10−6–10−7 M, hardly any effect could be detected, whereas consistently at concentrations around 10−8 M, a significant decrease was again observed. The phosphoprotein bands affected by in vitro addition of ACTH1–24 were of a smaller molecular weight than those affected by in vitro addition of cAMP.

Affinity‐Purified Anti‐B‐50 Protein Antibody: Interference with the Function of the Phosphoprotein B‐50 in Synaptic Plasma Membranes

Abstract: Affinity‐purified anti‐B‐50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B‐50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B‐50 protein and its adrenocorticotropin‐sensitive protein kinase, obtained from rat brain. Anti‐B‐50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B‐50 protein isolated by an improved procedure. The purified antibodies reacted only with the B‐50 and B‐60 protein, a proteolysis derivative (of B‐50), as assessed by the sodium dodecyl sulfate‐gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B‐50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5‐diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4‐phosphate was not altered. Inhibition by ACTH 1–24 of the endogenous phosphorylation of B‐50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4‐phosphate to phosphatidylinositol 4,5‐diphosphate. These data support our hypothesis on the functional interaction of B‐50 protein and phosphatidylinositol 4‐phosphate kinase in rat brain membranes. The evidence shows that purified anti‐B‐50 protein antibodies can be used to probe specifically the function of B‐50 protein in membranes.

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

Abstract: The localization of the phosphoprotein B‐50 (molecular weight 48,000, isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B‐50 protein. Two‐dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an immunochemical detection method employing an anti‐B‐50 antiserum revealed the presence of B‐50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B‐50 in nervous tissue. In addition, the regional distribution of endogenous B‐50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B‐50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1–24 is discussed.

ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity

ACTH1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation.

Modulation of the activity of purified phosphatidylinositol 4-phosphate kinase by phosphorylated and dephosphorylated B-50 protein

To investigate the modulation of phosphatidylinositol 4-phosphate kinase activity by the degree of phosphorylation of the B-50 protein, the enzyme was purified from rat brain cytosol by ammonium sulphate precipitation and DEAE-cellulose column chromatography. Purified rat brain B-50 was phosphorylated with protein kinase C and dephosphorylated with alkaline phosphatase. Incubation of the semi-purified phosphatidylinositol 4-phosphate kinase with 1 microgram of the B-50 preparation enriched in the dephospho-form, resulted in a small reduction of phosphatidylinositol 4-phosphate kinase activity (-16%), whereas incubation with the phospho B-50 preparation inhibited the enzyme activity by 40%. The effect of exogenous B-50 was studied in the presence of 10 micrograms albumin to minimize aspecific protein-protein interactions. The present data on the effect of exogenous B-50 protein on phosphatidylinositol 4-phosphate kinase activity, further support our hypothesis that the phosphorylation state of B-50 may be a regulatory factor in phosphoinositide metabolism in rat brain.

Resolution of Rat Brain Synaptic Phosphoprotein B‐50 into Multiple Forms by Two‐Dimensional Electrophoresis: Evidence for Multisite Phosphorylation

Abstract: Phosphoprotein B‐50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat‐bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5–5.0). As visualized by two‐dimensional gel electrophoresis, B‐50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B‐50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B‐50 was in part due to a glycoprotein nature of B‐50 was studied extensively. However, none of the six different methods used revealed the presence of glyco‐moieties in B‐50.

ACTH sensitive protein kinase from rat brain membranes

—The protein kinase which in rat brain synaptosomal plasma membranes is responsible for the phosphorylation of a protein band B‐50 (MW 48, 000) was inhibited by the behaviorally active peptide ACTH1–24 and not stimulated by cAMP. Treatment with 0.5% Triton X‐100 in 75 mM‐KCl solubilized 15% of the total B‐50 protein kinase activity and preserved the sensitivity of the enzyme to ACTH1–24. The rate of endogenous phosphorylation of protein band B‐50 was different in intact SPM, solubilized fraction and residue. cAMP stimulated the endogenous phosphorylation of the solubilized fraction in a rather general manner. The solubilized membrane material also phosphorylated B‐50 proteins which were previously extracted from membranes. Column chromatography of the solubilized material over DEAE‐cellulose pointed to the presence of multiple protein kinase activities from rat brain synaptosomal plasma membranes, one of which was the ACTH‐sensitive B‐50 protein kinase.

Immunohistochemical localization of a phosphoprotein (B 50) isolated from rat brain synaptosomal plasma membranes

The endogenous phosphorylation in vitro of at least 5 protein bands of rat brain synaptosomal membranes (SPM) is inhibited by the behaviorally active peptide ACTH1-24. One of these proteins, the phosphoprotein band B-50, was isolated from rat brain SPM by SDS-slab gel electrophoresis. An antiserum to B-50 was raised in rabbits. The presence of antibodies to B-50 in the antiserum was demonstrated by immunoperoxidase staining of cryostat sections of a polyacrylamide gel containing the antigen. The production of antibodies was monitored by an indirect immunofluorescence technique using cryostat sections of quick-frozen rat cerebellum. Immunofluorescent staining of the molecular and granular layers was observed, whereas the white matter and the perikarya and Purkinje cells of the cerebellum were not stained. With the use of the peroxidase-antiperoxidase (PAP) method, the immunohistochemical localization of the antigen in the molecular and granular layer of the cerebellum was confirmed. Regions rich in neuropil, like the CA1, CA2, CA3, CA4 and dentate gyrus showed an intense immunostaining. Thus, in agreement with the synaptic origin of B-50, the antiserum reacted with tissue components present in brian regions in synaptic contacts.