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PMID: 9521123 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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.

Protein science : a publication of the Protein Society ·Vol. 7 ·No. 2 ·1998-02-00 ·Pages 457-69

Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D

Abstract

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta 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 Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) 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 rCalpha(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.

MeSH Terms
Amides/chemistry Amino Acid Sequence Animals Asparagine/chemistry Catalysis Cattle Chromatography, Liquid/methods Cyclic AMP-Dependent Protein Kinases/chemistry,genetics,metabolism Escherichia coli/genetics Mass Spectrometry/methods Molecular Sequence Data Rabbits Rats Recombinant Proteins/chemistry,genetics,metabolism Swine
Chemicals
Amides Recombinant Proteins Asparagine Cyclic AMP-Dependent Protein Kinases
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Jedrzejewski P T
Department of Central Spectroscopy, German Cancer Research Center, Heidelberg.
Girod A
Tholey A
König N
Thullner S
Kinzel V
Bossemeyer D
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Article Info
Journal
Protein science : a publication of the Protein Society
Abbr.
Protein Sci
ISSN
0961-8368
DOI
10.1002/pro.5560070227
Published
1998-02-00
Pages
457-69
Language
English
Region
United States
NLM ID
9211750
PMCID
PMC2143929
Subset
IM
External Links
PubMed source DOI: 10.1002/pro.5560070227 PMC Full Text View journal details
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