A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry

• Published in: Protein science Vol. 7; no. 2; pp. 457 - 469
• Main Authors: Jedrzejewski, Paul T., Girod, Andreas, Tholey, Andreas, König, Norbert, Thullner, Sandra, Kinzel, Volker, Bossemeyer, Dirk
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01-02-1998
• Subjects: Amides - chemistry; Amino Acid Sequence; Animals; Asparagine - chemistry; cAMP‐dependent protein kinase; Catalysis; Cattle; Chromatography, Liquid - methods; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - genetics; Cyclic AMP-Dependent Protein Kinases - metabolism; deamidation; Escherichia coli - genetics; isoaspartate; isoenzymes; Mass Spectrometry - methods; Molecular Sequence Data; myristate; Rabbits; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Swine
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560070227

The N‐terminal sequence myr‐Gly‐Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes C, Cβ, and O. By capillary LC‐MS and tandem MS, we show that, in approximately one third of the C and Cβ enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C‐subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from C and Cβ. Asn 2 deamidation does not occur during C‐subunit preparation and is absent in recombinant myristoylated Cα (rCα) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N‐terminal glycine, verified by the myristoylation negative phenotype of an rCa(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr‐Gly‐Asp sequence. Asp 2 seems to be required for the well‐characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr‐Gly‐Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.