A muscle-derived substrate-bound factor that promotes neurite outgrowth from neurons of the central and peripheral nervous systems.
The development and survival of spinal motor neurons depends upon muscle-derived trophic factors. Some circumstantial evidence suggested to us that the regulatory subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAMP-dPK)-type II might be involved in neuritic outgrowth from spinal neurons. In the present study, we tested a commercial preparation of cAMP-dPK for neurite-promoting activity. Commercial cAMP-dPK-type II from skeletal and cardiac muscles elicited a significant neurite outgrowth from cultured embryonic chicken neurons when the enzyme preparation was bound to polylysine-coated substrata; type I cAMP-dPK from skeletal muscle was ineffective. Neither cAMP-dPK-type I nor -type II had a significant effect on the survival of spinal neurons in culture. Type II cAMP-dPK also stimulated neurite outgrowth from chicken cerebral hemisphere neurons, dorsal root ganglionic neurons, ciliary ganglionic neurons, and rat sympathetic ganglionic neurons in culture. The neurite-promoting activity appears to reside in a contaminant of the preparation since neither the purified regulatory nor catalytic subunits of cAMP-dPK-type II had an effect on neurite outgrowth per se from cultured neurons and since neurite-promoting activity did not correlate with [3H]cAMP binding or cAMP-dependent kinase activity. The neurite-promoting protein was then partially purified from commercial cAMP-dPK-type II by gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DE-52 cellulose. Sodium dodecyl sulfate gel electrophoresis of the active protein peak revealed a major protein band (MW 50 kDa) and several minor bands (e.g., MW 200 kDa, 52 kDa, 45 kDa). Also, immunoblot analysis and immunoprecipitation revealed that the partially purified neurite-promoting protein was distinct from laminin, heparan sulfate proteoglycan, nerve growth factor, neural cell adhesion molecule, and fibronectin. Furthermore, the neurite-promoting activity was not diminished by treatment with heparinase nor was it bound to heparin conjugated to Sepharose. Our results demonstrate that a protein unrelated to laminin or its associated macromolecules and which copurifies with the type II cAMP-dPK of striated muscle stimulates neurite outgrowth from neurons of the central and peripheral nervous systems.
Oh
T H
TH
Department of Anatomy, University of Maryland, School of Medicine, Baltimore 21201.
Markelonis
G J
GJ
Dion
T L
TL
Hobbs
S L
SL
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D002642:Chick Embryo; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005726:Ganglia, Parasympathetic / Q000166:cytology; D005727:Ganglia, Spinal / Q000166:cytology; D005728:Ganglia, Sympathetic / Q000166:cytology; D007118:Immunoassay; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D009474:Neurons / Q000648:ultrastructure*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D051381:Rats
D011506:Proteins; D000242:Cyclic AMP; D011494:Protein Kinases