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Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages
J S Sanghera1, S L Weinstein, M Aluwalia
1Biomedical Research Centre and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
Abstract:
Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
Insights
Bacterial lipopolysaccharide (LPS) activates mitogen-activated protein kinases (MAPK) and stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) in macrophages. LPS predominantly activates MAPK-activated protein kinase 2 (MAPKAPK2) via p38, distinct from phorbol 12-myristate 13-acetate (PMA) activation.
Area of Science:
- Cellular and Molecular Immunology
- Signal Transduction Pathways
Background:
- Bacterial lipopolysaccharide (LPS) is a potent activator of immune cells, particularly macrophages.
- Mitogen-activated protein kinases (MAPK) and stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) are critical signaling molecules in cellular responses.
- Understanding LPS-induced signaling is crucial for deciphering inflammatory and immune responses.
Purpose of the Study:
- To investigate the activation profiles of MAPK, SAPK/JNK, and related kinases following LPS stimulation in macrophages.
- To elucidate the specific pathways involved in the activation of MAPK-activated protein kinase 2 (MAPKAPK2) by LPS.
- To compare the signaling effects of LPS with phorbol 12-myristate 13-acetate (PMA).
Main Methods:
- Stimulation of murine macrophages (RAW 264.7) with bacterial LPS and PMA.
- Assessing kinase activity through phosphotransferase assays using substrates like myelin basic protein (MBP), GST-c-Jun, and hsp27.
- Utilizing MonoQ fractionation and immunoblotting to identify specific kinases (MAPK, SAPK/JNK, MAPKAPK2, p38 HOG1).
- Measuring tyrosine phosphorylation of p38 HOG1 and in vitro kinase assays for SAPK/JNK activity.
Main Results:
- LPS rapidly increased tyrosine phosphorylation and activation of MAPK, SAPK/JNK, and p38 HOG1.
- MAPKAPK2 activation by LPS occurred predominantly via p38 HOG1, unlike PMA which primarily activated MAPK.
- LPS, but not PMA, increased tyrosine phosphorylation of p38 HOG1, enhancing its kinase activity.
- SAPK/JNK activity increased 5- to 10-fold upon LPS treatment, with upstream kinases SEK1 and MEKK1 also activated.
Conclusions:
- LPS induces distinct activation patterns of MAPK family kinases in macrophages.
- The p38 HOG1 pathway plays a dominant role in LPS-mediated MAPKAPK2 activation.
- LPS and PMA differentially regulate MAPK signaling cascades, highlighting pathway specificity in immune cell activation.