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

Abstract

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