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

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylat ed cAPK (protein kinase A) catalytic subunit isozymes C alpha, C beta, and C gamma. By capillary LC-MS and tandem MS, we show that, in appro ximately one third of the C alpha and C beta enzyme populations from c attle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to As p 2. This deamidation accounts for the major isoelectric variants of t he cAPK C-subunits formerly called C-A and C-B Deamidation also includ es characteristic isoaspartate isomeric peptides from C alpha and C be ta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated C alpha (rC alpha) from Escheri chia coli. Deamidation appears to be the exclusive pathway for introdu ction of an acidic residue adjacent to the myristoylated N-terminal gl ycine, verified by the myristoylation negative phenotype of an rC alph a(Asn 2 Asp) mutant. This is the first report thus fas of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the wel l-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a con served site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially signif icant in view of the recent knowledge that negative charges close to m yristic acid in some proteins contribute to regulating their cellular localization.