Identification and expression analysis of three novel splice variants of protein kinase A catalytic β subunit gene in the mouse using combinatorial in silico and molecular biology approaches.
ABSTRACT The murine, bovine and human protein kinase A catalytic β (Cβ) subunit genes encode several splice variants. Ten human Cβ gene splice variants, namely Cβ1, Cβ2, Cβ3, Cβ3b, Cβ3ab, Cβ3abc, Cβ4, Cβ4b, Cβ4ab and Cβ4abc, have been reported. Four splice variants of the murine Cβ gene are homologues of human Cβ1, Cβ2, Cβ3 and Cβ4 variants. Using a combinatorial approach comprising bioinformatics tools and molecular biology techniques, we have identified three novel alternatively spliced transcript variants of the mouse Cβ gene designated Cβ5, Cβ6 and Cβ7. These transcript variants differ in their first coding exon. New exons of Cβ5 and Cβ6 variants can encode different N-terminals; however, the new exon of variant Cβ7, when spliced with exon 2, encountered a stop codon in all three reading frames and thus cannot form a functional protein. The Cβ6 variant showed an ubiquitous pattern of expression, whereas Cβ5 was not expressed in muscle tissue on postnatal days (PN)3 and 15. Also, Cβ7 was found to be expressed only in brain and muscle tissues at PN3 and was absent in all tissues examined at PN15 and PN60. The post-translational modification analysis showed characteristic signature sequence motifs in predicted proteins important for the functionality of the protein. The human homologues of variants reported in the present study have not yet been identified. The present study reports three novel splice variants that have not been identified using conventional approaches of alternative splice variant detection methods. Therefore, the approach used in the present study can be used to identify splice variants of genes in organisms. Database GenBank accession numbers: JN189786, JN189787 and JN189788.
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ABSTRACT: The unusual NH2-terminal blocking group of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein was found to be amide-linked n-tetradecanoic acid by gas chromatographic-, direct chemical ionization-, and fast atom bombardment-mass spectrometry. In addition, fast atom bombardment mass spectrometry revealed the presence of an additional alanine which had been overlooked when the original sequence was determined. The corrected and completed NH2-terminal sequence of the 350-amino acid catalytic subunit is CH3(CH2)12CONH-Gly-Asn-Ala-Ala-Ala-Ala-Lys.Proceedings of the National Academy of Sciences 11/1982; 79(20):6128-31. · 9.74 Impact Factor
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ABSTRACT: Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit. The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme. All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme. In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme. Its thermal stability is now comparable to the mammalian enzyme. All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex. Each shows an increase in T1/2 of 10 degrees C. This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability.Journal of Biological Chemistry 03/1993; 268(4):2348-52. · 4.65 Impact Factor
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ABSTRACT: We have constructed a database of alternatively spliced protein forms (ASP), consisting of 13,384 protein isoform sequences of 4422 human genes (www.bioinformatics.ucla.edu/ASP). We identified fifty protein domain types that were selectively removed by alternative splicing at much higher frequencies than average (p-value < 0.01). These include many well-known protein-interaction domains (e.g., KRAB; ankyrin repeats; Kelch) including some that have been previously shown to be regulated functionally by alternative splicing (e.g., collagen domain). We present a number of novel examples (Kruppel transcription factors; Pbx2; Enc1) from the ASP database, illustrating how this pattern of alternative splicing changes the structure of a biological pathway, by redirecting protein interaction networks at key switch points. Our bioinformatics analysis indicates that a major impact of alternative splicing is removal of protein-protein interaction domains that mediate key linkages in protein interaction networks. ASP expands the available dataset of human alternatively spliced protein forms from 1989 human genes (SwissProt release 42) to 5413 (nonredundant set, ASP + SwissProt), a nearly 3-fold increase. ASP will enhance the existing pool of protein sequences that are searched by mass spectroscopy software during the identification of peptide fragments.Journal of Proteome Research 01/2004; 3(1):76-83. · 5.06 Impact Factor