Complete primary structure and phosphorylation site of the 65-kDa macrophage protein phosphorylated by stimulation with bacterial lipopolysaccharide.
There is ample evidence that intracellular protein phosphorylation is a mandatory event in the process of macrophage activation by LPS, yet how this event is initiated and what roles the phosphorylated proteins are assigned to are poorly understood. We previously isolated a 65-kDa cytosolic protein (pp65) that was phosphorylated specifically in LPS-stimulated murine macrophages. In the present study, the complete primary structure of pp65 was determined on the basis of the cDNA containing an open reading frame of 1881 bases. The sequence of pp65 revealed that it is a murine homologue of human L-plastin, recently identified as a novel transformation-induced polypeptide of neoplastic human cells, and that it contains a unique series of Ca2+, calmodulin, and actin binding domains. A single phosphorylated peptide was isolated from the tryptic digest of pp65 by reverse-phase HPLC. From the amino acid sequence of the dodecapeptide Gly-Ser-Val-Ser-Asp-Glu-Glu-Met-Met-Glu-Leu-Arg, the phosphorylation site of pp65 was located at the N-terminal region adjacent to the first Ca2+ binding domain. This sequence contains a repeat of the casein kinase II motif Ser-Xxx-Xxx-Glu/Asp and, together with the preceeding Arg residue, constitutes the consensus sequence Arg-Xxx-Ser for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), but not mitogen-activated protein kinase (MAPK)-specific motif is found. These results, taken together with previous observations on the process of macrophage activation by LPS, demonstrate that pp65 is phosphorylated by an LPS-induced protein kinase other than MAPK and exerts its function on the cytoskeleton in a Ca2+/calmodulin-dependent manner.
Shinomiya
H
H
Department of Microbiology, Ehime University School of Medicine, Japan.
Hagi
A
A
Fukuzumi
M
M
Mizobuchi
M
M
Hirata
H
H
Utsumi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003598:Cytoskeletal Proteins; D004247:DNA / Q000737:chemistry / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000737:chemistry*; D008562:Membrane Glycoproteins; D051379:Mice; D008809:Mice, Inbred C3H; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid
D003598:Cytoskeletal Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; C026742:plastin; C066559:Lcp1 protein, mouse; D004247:DNA