Molecular cloning, sequence, and expression of a cDNA encoding the chicken myristoylated alanine-rich C kinase substrate (MARCKS).
Little is known about the important cellular substrates for protein kinase C (PKC) and their function in the cellular processes influenced by this kinase. This paper describes the molecular characteristics of a prominent cellular substrate for PKC in chicken cells, known as the myristoylated alanine-rich C kinase substrate, or MARCKS protein. The chicken protein was studied because it was apparently at least 20 kilodalton smaller than its mammalian counterpart; we hoped that regions of sequence similarity might point to conserved regions of biological importance. Using the bovine MARCKS cDNA as a probe, we selected a positive clone from a chicken brain cDNA library that contained an insert of about 1.5 kilobase, in which a single open reading frame encoded a protein of 281 amino acids, 27.7 kilodaltons, pI 5.26. This protein contained the sequences of ten tryptic peptides derived from the purified chicken brain protein. Expression of the cDNA insert in mammalian cells confirmed that the open reading frame encoded a protein that comigrated on two-dimensional electrophoresis with the authentic chicken protein, and could be phosphorylated by exposure of the cells to active phorbol esters. When the chicken and bovine protein sequences were compared, the two major regions of sequence identity were: 1) the amino terminal region containing a myristoylation consensus sequence and an mRNA splice site, and 2) a highly basic internal domain of 25 amino acids that contained all of the serines known to be phosphorylated by PKC in the intact protein. These conserved regions are likely to represent domains of some functional importance for this widely distributed cellular substrate for PKC.
Graff
J M
JM
Howard Hughes Medical Institute Laboratories, Durham, North Carolina 27710.
Stumpo
D J
DJ
Blackshear
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002645:Chickens / Q000235:genetics; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D004247:DNA; D013755:Tetradecanoylphorbol Acetate