The aim of this endeavor was to isolate and characterize, through cDNA cloning, the various subunits of cyclic AMP-dependent protein kinase (A kinase) present in the nervous system of the marine mollusc Aplysia californica and to determine how these proteins participate in neuronal modulation. The current work focuses on the isolation and characterization of four related catalytic (C) subunits of A kinase that arise via alternative splicing of mRNA transcribed from a single gene by comparison to the mammalian isoforms which are transcribed from independent genes. Unique amino acid sequences present in the alternatively expressed axons make it possible to produce axon-specific probes to discriminate between isoforms at the nucleic acid and protein levels. Antibodies have already been generated that distinguish between proteins having alternate N-termini. Using these tools, we intend to investigate how C subunits respond to neuromodulators that act through the A kinase cascade. In addition to these isoforms, a fifth, more distantly related isoform was cloned and found to be exclusively expressed in sperm. This kinase has approximately 58% amino acid homology with the other C subunits, and localizes by EM immunocytochemistry to the base of the axoneme in mature spermatozoa. Efforts are currently underway to determine if this kinase contributes to the acquisition of sperm motility or participates in fertilization.
