Muscarinic receptor activation stimulates B-50/GAP43 phosphorylation in isolated nerve growth cones.

A characteristic feature of neurite formation is high expression of the phosphoprotein B-50/GAP43. Previous studies with growth cone membranes have indicated that this neuron-specific protein kinase C substrate may be involved in transmembrane signal transduction at the growth cone. We monitored the degree of phosphorylation of B-50 by quantitative B-50 immunoprecipitation from intact nerve growth cones, isolated from 5-day-old rat brain and prelabeled with 32P-orthophosphate. B-50 phosphorylation in nerve growth cones is stimulated by 4 beta-phorbol 12,13-dibutyrate (PDB) and 1,2-dioctanoylglycerol (DOG) in a concentration-dependent manner, but not by 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD). These results confirm that B-50 is a substrate of PKC in intact growth cones. Depolarization induced by 30 mM K+ produces a transient increase in B-50 phosphorylation, which is maximal after 15 sec and declines to basal level within 5 min. This rise in B-50 phosphorylation can be partially blocked by atropine (10(-3)-10(-4) M), suggesting the involvement of muscarinic receptors. Indeed, the cholinergic receptor agonist carbachol enhances B-50 phosphorylation in a concentration-dependent manner (50% at 10(-3) M). Since the effect of carbachol (10(-3) M) can be blocked by atropine (10(-7) M), we conclude that this increase in B-50 phosphorylation is mediated through activation of the muscarinic receptors on the growth cones. The carbachol-induced stimulation is further increased by concurrent K+-depolarization. The effects of carbachol and depolarization are additive. To our knowledge, this is the first report showing receptor-mediated effects on the PKC substrate B-50 in growth cones. Our data support the hypothesis that phosphorylation of B-50 by PKC is involved in signal transduction in nerve growth cones.

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.

Role of the growth cone in neuronal differentiation.

Nerve growth cones are motile, exploring organelles at the tip of a growing neurite. The growth cone is a highly specialized structure, equipped with a complex machinery for reversible membrane expansion and rapid cytoskeletal reorganization, a machinery required for growth cone motility and neurite elongation. It also contains perception systems that enable the growth cone to respond to external signals, thereby steering the trailing neurite to the correct target. Soluble and substrate bound guidance molecules in the environment modulate growth cone behavior either through direct interaction or classical receptor activation coupled to second messengers. A prominent phosphoprotein of the growth cone is B-50. We propose a role for this growth-associated protein kinase C substrate in signal transduction processes in the growth cone.

In vivo and in vitro studies in a 46, XY phenotypically female infant with 17-ketosteroid reductase deficiency.

A 46, XY phenotypically female infant with 17-ketosteroid reductase (17-KSR) showed normal plasma androgens for chromosomal sex shortly after birth, but did not show the physiologic testosterone rise. One intramuscular injection with human chorionic gonadotropin resulted in high ratios between androstenedione/testosterone and dehydroepiandrosterone/delta 5-androstenediol, confirming the diagnosis. In spermatic vein plasma similarly elevated ratios were found. A urinary steroid profile revealed elevated levels of metabolites of 17-OH-progesterone and androstenedione. In vitro studies in testicular tissue showed a decreased capacity of 17-ketosteroid reductase, the reduction capacity being more affected than the oxidation capacity. The activity of 3 beta-hydroxysteroid-dehydrogenase was slightly increased. The serial analysis of plasma androgens provides more insight in the natural history of 17-ketosteroid reductase.

B-50 phosphorylation and polyphosphoinositide metabolism in nerve growth cone membranes.

The neuron-specific phosphoprotein B-50 (Mr 48 kDa, isoelectric point, IEP, 4.5), which is identical to GAP43, is a member of a family of growth-associated proteins. Protein B-50 is a major phosphoprotein in nerve growth cones isolated from fetal rat brain. In a growth cone particulate fraction (GCp), endogenous B-50 phosphorylation is Ca2+-dependent and is unaffected by cAMP. Addition of purified protein kinase C (PKC) to GCP enhances B-50 phosphorylation. In heat-inactivated GCp, B-50 is one of the major substrates of purified PKC. Endogenous B-50 phosphorylation in GCP is stimulated in a dose-dependent manner by 4 beta-phorbol diesters, known to activate PKC, but not by the inactive 4 alpha-phorbol derivatives. In synaptic plasma membranes (SPM) isolated from adult rat brain, the degree of B-50 phosphorylation has been implicated in the modulation of receptor-mediated polyphosphoinositide (PPI) hydrolysis. In addition to B-50 and its kinase, PKC, the GCp fraction was also shown to contain all other components of such a modulatory system: the phosphatidylinositol 4-phosphate (PIP)-kinase, as shown on Western blots with affinity-purified IgGs against PIP-kinase, and the polyphosphoinosides, PIP and phosphatidylinositol 4,5-bisphosphate (PIP2), since the addition of gamma-32P-ATP to the GCp fraction not only results in B-50 phosphorylation but also in the labeling of phosphatidic acid (PA), PIP, and PIP2. ACTH1-24, which inhibits B-50 phosphorylation in the GCp fraction in a dose-dependent manner (IC50 = 5 x 10(-6) M), stimulates PIP2 labeling dose-dependently in the same preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinase C substrate protein B-50 and axonal regeneration.

As reported previously the prominent protein kinase C substrate protein B-50 is present in growth cones isolated from fetal rat brain and in outgrowing hippocampal neurites. These findings suggest that B-50 plays a role in axonal growth during development of the nervous system. In the present paper the fate of B-50 is investigated in regenerating rat sciatic nerve. Using affinity-purified anti-B-50 antibodies B-50 levels have been compared in crushed and contralateral intact nerves by means of immunoblotting and radioimmunoassay. B-50 levels in the crushed nerve increased 5.3-fold as compared to non-crushed controls. Furthermore, the cellular localization of B-50 has been assessed by immunohistochemistry. Virtually no B-50 immunoreactivity was seen in control nerves, but bright immunofluorescence appeared in regenerating sprouts. Our data are in line with current evidence from several laboratories that B-50 is a member of a small family of growth-associated proteins and support the hypothesis that B-50 is involved in axonal growth.

Nerve growth factor enhances the level of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.

Exposure of PC12 cells to nerve growth factor results in arrest of cell growth and induction of differentiation to sympathetic neuron-like cells, bearing neurites. In this study we identify a 48 kDa PC12 phosphoprotein as the neuron-specific protein kinase C substrate B-50 (Mr 48 kDa; IEP 4.5) on basis of comigration with purified B-50, immunoreactivity and phosphopeptide mapping. B-50 is present in both undifferentiated and differentiated PC12 cells. Exposure of PC12 cells to nerve growth factor for two days results in a 2.5-fold increase in the amount of B-50 as measured by RIA. Indirect immunofluorescence microscopy reveals that B-50 is mainly localized at the cell membrane and in growth cones. Our data are in line with the hypothesis that B-50 plays a role in neurite outgrowth and indicate that PC12 cells provide a suitable model to study this hypothesis.

Phosphoprotein B-50 in nerve growth cones from fetal rat brain.

The presynaptic, nervous tissue-specific phosphoprotein B-50 is present in infant and adult rat brain. In the present study we demonstrate that B-50 is a major phosphoprotein in nerve growth cones obtained from fetal rat brain. As this protein is an endogenous substrate for protein kinase C, an enzyme linked to cell growth and proliferation, a role for B-50 in nerve growth cone function is suggested.

Mitogenic stimulation of malignant B cells. Chronic lymphocytic leukaemia: secretion of monoclonal IgM by in vitro-induced plasmablasts.

This study provides evidence that leukaemic B cells are able to proliferate and differentiate into plasmablasts. The Ig produced was monoclonal in nature and this was shown by gel electrophoresis.