RESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clonagem Molecular , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Imunoprecipitação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Peptídeos/síntese química , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TrítioRESUMO
The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of many aspects of eukaryotic cellular activity. In the free-living nematode, Caenorhabditis elegans, two genes encode PK-A-like catalytic subunits. The kin-1 gene has the potential to generate, through alternative splicing events, a multiplicity of catalytic subunit isoforms; in contrast, the F47F2.1b gene appears to encode just a single authentic catalytic subunit-like protein. Here, we report on the occurrence of, and developmental changes in the expression of, polypeptide products of these genes in both C. elegans and the closely related nematode, C. briggsae. Polypeptides derived from the F47F2.1 gene and its orthologue were detected in mixed stage populations of C. elegans and C. briggsae, respectively. Likewise, a number of polypeptides arising as a result of alternative splicing of transcripts from kin-1, or its orthologue in C. briggsae, were identified in mixed stage populations of nematodes. These isoforms included polypeptides with N-termini encoded by exons N'1 or N'4 and C-termini encoded by exons 7 or N. The expression of isoforms with an N-terminus encoded by the N'1 exon is of significance because the amino acid sequence encoded by this exon encompasses an N-myristoylation motif. Isoform abundance appears to be related to developmental stage. Substantial differences in polypeptide expression profiles can be seen in embryonic and adult nematodes. The functional significance of this PK-A catalytic subunit isoform diversity is discussed.
