Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide.
Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.
Shinomiya
H
H
Department of Microbiology, Jichi Medical School, Tochigi-Ken, Japan.
Hirata
H
H
Nakano
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003600:Cytosol / Q000737:chemistry; D007375:Interleukin-1 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D008809:Mice, Inbred C3H; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000502:physiology; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism
D000596:Amino Acids; D007375:Interleukin-1; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D011506:Proteins