Characterisation of the N′1 isoform of the cyclic AMP-dependent protein kinase (PK-A) catalytic subunit in the nematode, Caenorhabditis elegans

• Published in: Archives of biochemistry and biophysics Vol. 519; no. 1; pp. 38 - 45
• Main Authors: Clegg, Roger A., Bowen, Laura C., Bicknell, Annalise V., Tabish, Mohammad, Prescott, Mark C., Rees, Huw H., Fisher, Michael J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.03.2012
• Subjects: Alternative splicing; Isoform; N-myristoylation; Nematode; Recombinant; N-myristoylation; Alternative splicing; Isoform; Nematode; Recombinant
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2012.01.008

► N′1-isoforms of Caenorhabditis elegans PK-A catalytic subunits are N-myristoylatable. ► The presence of myristoyl–glycine has been confirmed by MALDI-TOF MS. ► Protein with C. elegans N-terminus and murine C-terminus has been prepared. ► Myristoylation has little effect on the catalytic properties of the chimera. ► Myristoylated chimera has greater apolar targeting than murine protein. 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 [3H]-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.