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Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with
H Shinomiya1, H Hirata, M Nakano
1Department of Microbiology, Jichi Medical School, Tochigi-Ken, Japan.
Abstract:
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.
Insights
Bacterial lipopolysaccharide (LPS) triggers protein phosphorylation in macrophages, leading to the identification of a novel 65 kDa protein (pp65). This pp65 phosphorylation is crucial for macrophage activation and inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Protein phosphorylation is a key regulator of cellular responses to external stimuli.
- Bacterial lipopolysaccharide (LPS) is a potent activator of macrophages, initiating complex intracellular signaling cascades.
Purpose of the Study:
- To identify and characterize novel phosphorylated proteins induced by LPS in macrophages.
- To investigate the role of these phosphorylated proteins in LPS-mediated macrophage activation.
Main Methods:
- Murine peritoneal macrophages were stimulated with LPS.
- Phosphorylated proteins were analyzed using SDS-PAGE and autoradiography.
- A 65 kDa phosphorylated protein (pp65) was purified using sequential chromatography.
- Western blot analysis was performed using anti-pp65 antibodies.
- LPS-nonresponsive C3H/HeJ macrophages were used for comparative studies.
Main Results:
- LPS induced a distinct set of phosphorylated proteins (pp) in macrophage cytosol and membrane fractions.
- A novel 65 kDa phosphorylated protein (pp65) was purified, exclusively phosphorylated on serine residues.
- LPS-induced pp65 phosphorylation was absent in C3H/HeJ macrophages, indicating a defect upstream of phosphorylation.
- pp65 phosphorylation levels correlated with cellular responses like IL-1 production.
Conclusions:
- pp65 is a novel, LPS-inducible phosphorylated protein crucial for macrophage activation.
- The phosphorylation of pp65 is mediated by an LPS-inducible serine kinase.
- Defects in LPS responsiveness in C3H/HeJ mice occur before pp65 phosphorylation.
- pp65 plays a significant role in LPS-induced macrophage activation and inflammatory signaling.