Protein phosphorylation during afterdischarge in peptidergic neurons of Aplysia.

The bag cells (BC) of Aplysia are a group of peptidergic neurons that respond to brief electrical stimulation by producing a prolonged afterdischarge of synchronous action potentials. We have demonstrated previously that the cyclic nucleotide, cyclic 3’:5’-adenosine monophosphate (cyclic AMP), plays a role in the genesis of the bag cell afterdischarge and that the microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase increases the excitability of bag cell neurons in culture. In this paper, we present evidence that the phosphorylation state of at least two proteins (proteins BC-1 and BC-2) are enhanced after the onset of afterdischarge. One of these proteins, BC-2 (approximately 21,000 daltons), undergoes cyclic AMP-stimulated phosphorylation in cell-free extracts in vitro and is the major substrate in bag cell tissues for exogenous cyclic AMP-dependent protein kinase catalytic subunit. This protein undergoes a substantial change in phosphorylation at 20 min into the afterdischarge using both bag cells prelabeled with Na2H”‘P04 and a “back phosphorylation” (postlabeling) procedure. No change could, however, be detected early (2 min) in the afterdischarge with either technique for this protein. We have determined that BC-2 phosphoprotein is apparently specific to bag cell tissue and is not the precursor to the bag cell neurosecretory peptide previously described. We also have determined the amino acid composition and present a partial sequence of the NH2 terminus obtained from 100 pmol of protein eluted from a sodium dodecyl sulfate-polyacrylamide gel band. Using the method of prelabeling phosphoproteins in bag cells with Na2H”“P04, we observed an 82% increase in phosphorylation of a 33,000-dalton protein (BC-1) at 2 min and a 69% increase at 20 min into the afterdischarge. In vitro labeling with [Y-~P]ATP (adenosine triphosphate) demonstrated that this protein undergoes cyclic AMP-dependent phosphorylation and that its phosphorylation also was enhanced markedly by exogenous cyclic AMP-dependent protein kinase. BC1 does not appear to be a phosphoprotein unique to bag cells and, from Coomassie blue staining, is a minor component in this tissue. We have described two proteins in bag cell tissues whose phosphorylation state is altered by both cyclic AMP with in vitro labeling and by afterdischarge in the intact bag cell cluster. A change in the phosphorylation state of these proteins may be related to the electrical events of afterdischarge or to its biochemical sequelae. Many neuronal and neuroendocrine cells undergo exzymes which leads to the phosphorylation of specific citability changes that are thought to involve cyclic nuprotein substrates. Changes in the state of protein phoscleotides or calcium ions. In eucaryotic cells, the only phorylation have been hypothesized to be responsible for known means by which cyclic nucleotides act is by stimaltering the ionic conductances and, hence, the state of ulating the activity of endogenous protein kinase enexcitability of individual nerve cells (Greengard, 1978). It has been shown previously that the cyclic nucleotide, ’ We would like to thank Dr. M. Hunkapiller for performing the electroelution of the BC-2 protein from the polyacrylamide gel. We would also like to thank Dr. M. Kennedy and Dr. A. Chiu for their ’ Present address: Department of Pharmacology, Yale University comments on an earlier version of the manuscript. The research reSchool of Medicine, New Haven, CT 06510. ported in this paper was supported by National Institutes of Health ,’ Present address: The Genetics Institute, 225 Longwood Avenue, Grants NS 13896 and 15183 to F. S. K. R. J. is a Gordon Ross Medical Boston, MA 02115. Foundation Fellow. 4 To whom correspondence should be addressed.