Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo.
Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.
Jose
Antony M
AM
Department of Molecular, Cellular, and Developmental Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Koelle
Michael R
MR
D000483:Alleles; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002460:Cell Line; D003062:Codon; D018388:Codon, Terminator; D019852:Diacylglycerol Kinase / Q000737:chemistry*; D004075:Diglycerides / Q000737:chemistry*; D005091:Exons; D006801:Humans; D007313:Insecta; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D003062:Codon; D018388:Codon, Terminator; D004075:Diglycerides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019852:Diacylglycerol Kinase