Presence of conserved domains in the C-terminus of MARCKS, a major in vivo substrate of protein kinase C: application of ion trap mass spectrometry to the elucidation of protein structures.
MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously analyzed MARCKS purified from bovine brain using capillary liquid chromatography/electrospray mass spectrometry and found that the protein structure differed significantly from that deduced from cDNA sequences [Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302]. Moreover, the alignment of the protein from various species showed a lack of any conserved sequences in the C-terminal half of the molecule. This prompted us to reexamine the C-terminal amino-acid sequence of bovine MARCKS. The purified protein was digested with lysyl endoprotease, and the obtained C-terminal peptide was further digested with either Staphylococcus V8 protease or NTCB. The small peptides thus obtained were analyzed by liquid chromatography/electrospray/tandem mass spectrometry. This combined with gas-phase Edman sequencing allowed us to determine the C-terminal primary structure. The sequence obtained differed significantly from that reported previously, and the comparison with other species revealed the presence of a novel conserved domain in the C-terminal region of MARCKS.
Yamauchi
E
E
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603.
Kiyonami
R
R
Kanai
M
M
Taniguchi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D017124:Conserved Sequence; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents; D013861:Thiocyanates
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; D013439:Sulfhydryl Reagents; D013861:Thiocyanates; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; C008658:2-nitro-5-thiocyanobenzoic acid