Evolutionary conservation of the signaling proteins upstream of cyclic AMP-dependent kinase and protein kinase C in gastropod mollusks

Abstract

The protein kinase C (PKC) and the cAMP-dependent kinase (protein kinase A; PKA) pathways are known to play important roles in behavioral plasticity and learning in the nervous systems of a wide variety of species across phyla. We briefly review the members of the PKC and PKA family and focus on the evolution of the immediate upstream activators of PKC and PKA i.e., phospholipase C (PLC) and adenylyl cyclase (AC), and their conservation in gastropod mollusks, taking advantage of the recent assembly of the Aplysiacalifornica and Lottia gigantea genomes. The diversity of PLC and AC family members present in mollusks suggests a multitude of possible mechanisms to activate PKA and PKC; we briefly discuss the relevance of these pathways to the known physiological activation of these kinases in Aplysia neurons during plasticity and learning. These multiple mechanisms of activation provide the gastropod nervous system with tremendous flexibility for implementing neuromodulatory responses to both neuronal activity and extracellular signals.

Fig. 1

Alignment of the last exon in classical PKCs from a number of organisms showing the differential splicing in mollusks and vertebrates. Shaded residues are conserved in all species. Residues in green are specifically conserved between the nervous system exon in Aplysia, the orthologous exon in Lottia and vertebrate PKCβ1, but not conserved in the alternative exon. The * signifies conserved phosphorylation sites in PKCs.

Fig. 2

Results of a Phylip analysis of merged X and Y domains of PLCs from different species. Six different families were identified, shown on the right. For the vertebrates, human (Hom) and Danio (DAN), the accepted name of the PLC is given. For the primitive chordates, Branchiostoma (BRA) and Ciona (CIO), the protein IDs are given from the NCBI entry. For Apis (API) and Capitella (CAP), the protein IDs are given from the NCBI and JGI genome sites, respectively, and for Drosophila (DRO), the accepted gene names are used. For the mollusks the names are blue. For Aplysia (APL) the contig from the NCBI site containing the X and Y domains is given. For Lottia (LOT) the protein IDs are given from the JGI genome site. The numbers represent the percentage of trees generated by the Phylip protein Neighbour that contained the tree shown. Only selected branches are highlighted to emphasize the division into PLC families. The outgroup (not shown) was yeast (Schizosaccharomyces pombe) PLC.

Fig. 3

Results of a ClustalW analysis of highly conserved sequences within the catalytic domain of the C2A region of ACs. Residues highlighted in yellow represent amino acids conserved in 90% or more of the AC isoforms. Residues highlighted in grey represent conserved substitutions. Lysine and aspartate residues indicted in green distinguish adenylyl cyclases from guanylyl cyclases [Baker and Kelly, 2004]. The glycine residue in the Drosophila rutabaga AC highlighted in purple is Gly1026, which is altered to Arg in the rutabaga learning mutant, resulting in complete loss of catalytic activity [Levin et al., 1992]. For human (Hom), standard nomenclature is used; and for corresponding Danio (DAN) isoforms, the same nomenclature is employed. For Drosophila (DRO), gene names are used. For Aplysia (Apl) ACs, names are from NCBI and Lin et al. [in preparation]. For Lottia (Lot) isoforms that correspond to Aplysia isoforms, the same nomenclature is used. For C. elegans (Cel), protein IDs are from NCBI. For Capitella (Cap) and for one Lottia AC, names correspond to protein IDs from the JGI Genome site. For Drosophila, two of the members of the ACX family, ACXA and ACXC, have been omitted from the analysis, as they are extremely similar to ACXB. To facilitate comparison with the genome sites, AC IDs that are somewhat different from those in the databases are listed below. C. elegans: NP_504553; NP_504486, NP_497970. Danio AC1 = XP_685077; Danio AC2 iso A = XP_692173; Danio AC2 iso B = NP_001093457; Danio AC3 iso A = XP_700547; Danio AC3 iso B = XP_693142; Danio AC6 = XP_001922749; Danio AC7 = XP_001922494; Danio AC8 = NP_001137224; Drosophila ACXD = ACK77593; Lottia A = 141290; Lottia B = 187968/231713; Lottia C = 135065; Lottia D = 136240/170789. (For Lottia B and D, on JGI, the C1A and C2A domains were given two separate proteins numbers.)

Fig. 4

Results of a Phylip analysis of the C2A domains of AC from different species. For the vertebrates, human (Hom) and Danio (DAN), the accepted name of the AC is given. For C. elegans (CEL), the gene numbers from the NCBI genome site are used and for Drosophila (DRO), the accepted gene names are used, for Capitella (CAP) the protein IDs are given from the JGI genome site. For the mollusks the names are blue. For Aplysia (APL) the name from Lin et al. [in preparation] is used. For Lottia (LOT), the same designations are used as for Aplysia when the ACs correspond to the Aplysia ACs; for the fifth AC from Lottia, the protein ID from the JGI genome site is used. Further explanation of the protein IDs is included in the legend for figure 3. The numbers represent the percentage of trees generated by the Phylip protein Neighbour that contained the tree shown. Only selected branches are highlighted to emphasize the division into AC families. Dictyostelium ACA (not shown) served as the outgroup for this analysis.