Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: content and phosphorylation of B-50.
The content and phosphorylation of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B-50 immunolocalization in the same cultures. In control cultures, the concentration of B-50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B-50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B-50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B-50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B-50 and NSE after 14 DIV. The basal phosphorylation of B-50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B-50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B-50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B-50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.
Ramakers
G J
GJ
Netherlands Institute for Brain Research, Amsterdam.
De Graan
P N
PN
Oestreicher
A B
AB
Boer
G J
GJ
Corner
M A
MA
Gispen
W H
WH
D000818:Animals; D001345:Autoradiography; D002540:Cerebral Cortex / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004594:Electrophysiology; D005260:Female; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013779:Tetrodotoxin / Q000494:pharmacology
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D013779:Tetrodotoxin; D004247:DNA; D011493:Protein Kinase C; D010751:Phosphopyruvate Hydratase