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Association of mitogen-activated protein kinases with microtubules in mouse macrophages
1Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York 10021, USA.
Abstract:
Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole-disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule-associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.
Insights
Lipopolysaccharide (LPS) activates microtubule-associated protein kinases (MAPK) in macrophages. This study found that LPS preferentially activates MAPK already bound to microtubules, suggesting a key role for these microtubule-associated kinases in LPS signaling.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Lipopolysaccharide (LPS) triggers significant responses in mouse macrophages, with similar effects observed with the microtubule-binding agent Taxol.
- The genetic control for LPS and Taxol responses overlap, suggesting a role for microtubule-associated proteins (MAP) in LPS signaling pathways.
Purpose of the Study:
- To investigate the localization and activation of mitogen-activated protein kinases (MAPK) in macrophages stimulated by LPS.
- To determine if MAPK are associated with microtubules and if this association influences their activation by LPS.
Main Methods:
- Immunofluorescence microscopy to visualize MAPK localization within macrophage filaments.
- Co-isolation of MAPK with polymerized tubulin from macrophage and brain extracts.
- Fractionation of primary macrophages to isolate MAPK associated with different cellular components, including microtubules.
Main Results:
- A significant portion of the LPS-activatable MAPK pool in primary mouse peritoneal macrophages is associated with microtubules.
- MAPK were localized to cellular filaments disrupted by colchicine and nocodazole, indicating microtubule association.
- While LPS stimulation did not alter the proportion of MAPK bound to microtubules, it preferentially activated the microtubule-associated pool.
Conclusions:
- The findings support the hypothesis that LPS signaling in macrophages involves the activation of microtubule-associated kinases.
- This study identifies a novel pathway where LPS preferentially activates MAPK bound to microtubules, highlighting the role of microtubule-associated proteins in innate immune responses.