title: Characterisation of the N'1 isoform of the cyclic AMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans.
abstract: Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N'3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N'1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N'1 C-subunit isoforms, following incubation of nematodes with [(3)H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl-glycine in the C-subunit. In order to better understand the impact of the N'1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N'1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N'1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit.
authors.affiliation: Hannah Research Institute, Ayr KA6 5HL, Scotland, UK.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Clegg
authors.forename: Laura C
authors.initials: LC
authors.lastname: Bowen
authors.forename: Annalise V
authors.initials: AV
authors.lastname: Bicknell
authors.forename: Mohammad
authors.initials: M
authors.lastname: Tabish
authors.forename: Mark C
authors.initials: MC
authors.lastname: Prescott
authors.forename: Huw H
authors.initials: HH
authors.lastname: Rees
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Fisher
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014316:Tritium
chemical_list: D029742:Caenorhabditis elegans Proteins; D007527:Isoenzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D014316:Tritium; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits