Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide.
We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alanine-rich C kinase substrate). The murine MARCKS cDNA clone encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SDS/PAGE with an apparent molecular mass of 68 kDa. Peptide mapping studies indicated that MARCKS produced by the transfected gene was indistinguishable from the endogenous murine macrophage protein. Comparison of the murine macrophage sequence with the previously published chicken and bovine brain sequences revealed two conserved domains: an N-terminal membrane-binding domain and a phosphorylation domain that also contains calmodulin and actin binding sites. In murine peritoneal macrophages, bacterial lipopolysaccharide increased MARCKS mRNA levels by greater than 30-fold. Multiple MARCKS transcripts were observed and could be accounted for by differential polyadenylylation and incomplete processing. Genomic Southern blot analysis suggested a single MARCKS gene per haploid genome.
Seykora
J T
JT
Rockefeller University, New York, NY 10021.
Ravetch
J V
JV
Aderem
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D015723:Gene Library; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D004247:DNA; D011493:Protein Kinase C