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The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

ABSTRACT: The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC.

SUBMITTER: Schober JM

PROVIDER: S-EPMC3259197 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

Schober Joseph M JM Kwon Guim G Jayne Debbie D Cain Jeanine M JM

Biochemical and biophysical research communications 20111119 1

The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were deple ...[more]

PMID: 22120625

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Project description:Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.

Dataset Information

The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

Publications

The microtubule-associated protein EB1 maintains cell polarity through activation of protein kinase C.

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