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Endotoxin induces rapid protein tyrosine phosphorylation in 70Z/3 cells expressing CD14
1Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
CD14, a glycosylphosphatidylinositol-anchored glycoprotein of leukocytes, binds endotoxin (lipopolysaccharide (LPS)) with high affinity. After the murine pre-B cell line 70Z/3 is transfected with DNA encoding human CD14 (hCD14), the resultant stably transfected cell line, 70Z/3-hCD14, responds to 1000-fold lower LPS concentrations than the parental CD14-negative line. We have used 70Z/3-hCD14 cells, RAW264.7 cells, and elicited murine peritoneal exudate macrophages (PEM) to study LPS-induced protein tyrosine phosphorylation. LPS induces the rapid tyrosine phosphorylation of a 38-kDa protein (p38) in 70Z/3-hCD14 cells, PEM, and RAW264.7 cells and of two isoforms of mitogen-activated protein kinases (MAPK) in only RAW264.7 cells and PEM. p38 can be distinguished from the MAPK isoforms based on differences in mobilities on SDS-polyacrylamide gel electrophoresis and the lack of reactivity of p38 with anti-MAPK antibody even after dephosphorylation with potato acid phosphatase. Synthetic lipid A induces p38 phosphorylation in 70Z/3-hCD14 cells, whereas phorbol 12-myristate 13-acetate and interferon-gamma fail to induce tyrosine phosphorylation of p38. Pretreatment of 70Z/3-hCD14 cells with anti-hCD14 monoclonal antibody or the tyrosine kinase inhibitor herbimycin A inhibits LPS-induced tyrosine phosphorylation of p38. These results suggest that increased protein tyrosine phosphorylation occurs rapidly after LPS binds to CD14 and is likely to be an important event in mediating LPS-induced cell activation.
Insights
Lipopolysaccharide (LPS) binding to CD14 triggers rapid protein tyrosine phosphorylation, specifically of a 38-kDa protein (p38). This phosphorylation is crucial for LPS-induced immune cell activation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- CD14 is a leukocyte glycoprotein that binds lipopolysaccharide (LPS) with high affinity.
- Transfected cells expressing human CD14 (hCD14) show increased sensitivity to LPS.
- Understanding LPS-induced signaling pathways is critical for immune response research.
Purpose of the Study:
- To investigate LPS-induced protein tyrosine phosphorylation in cells expressing CD14.
- To identify key proteins involved in LPS-mediated signaling.
- To elucidate the role of CD14 in initiating tyrosine phosphorylation cascades.
Main Methods:
- Utilized a murine pre-B cell line (70Z/3) transfected with hCD14 (70Z/3-hCD14 cells).
- Employed RAW264.7 cells and elicited murine peritoneal exudate macrophages (PEM) for comparative analysis.
- Analyzed protein tyrosine phosphorylation using SDS-PAGE and Western blotting techniques, including treatment with specific inhibitors and antibodies.
Main Results:
- LPS rapidly induced tyrosine phosphorylation of a 38-kDa protein (p38) in 70Z/3-hCD14 cells, RAW264.7 cells, and PEM.
- Mitogen-activated protein kinases (MAPK) isoforms were phosphorylated only in RAW264.7 cells and PEM, distinct from p38.
- Phosphorylation of p38 was triggered by synthetic lipid A and inhibited by anti-hCD14 antibody and a tyrosine kinase inhibitor, herbimycin A.
Conclusions:
- LPS binding to CD14 initiates a rapid protein tyrosine phosphorylation cascade.
- The 38-kDa protein (p38) is a key substrate in LPS-induced signaling through CD14.
- Tyrosine phosphorylation following CD14 activation is a significant early event in LPS-mediated cell activation.