Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1.

Klebanovych Klebanovych,Dráberová Dráberová,Dráber Dráber,Sládková Sládková,Sulimenko Sulimenko,Vosecká Vosecká,Čapek Čapek

doi:10.3390/cells8040345

Abstract

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

Highlights

IntroductionMast cells play a crucial role in allergies, as well as in innate and adaptive immune responses
Mast cells play a crucial role in allergies, as well as in innate and adaptive immune responses.They express plasma membrane-associated high-affinity IgE receptors (FcεRIs), the aggregation of which by a multivalent antigen (Ag)-IgE complex triggers mast cell activation, resulting in their degranulation; in the release of inflammatory mediators, proteases, and lipid mediators; and in the production of various cytokines [1]
In the bone marrow-derived mast cells (BMMCs) line used in this study, the P-Tyr signal in Ag-activated cells substantially increased at the beginning of activation but gradually weakened to the original level (Figure 1A)

Summary

Introduction

Mast cells play a crucial role in allergies, as well as in innate and adaptive immune responses. They express plasma membrane-associated high-affinity IgE receptors (FcεRIs), the aggregation of which by a multivalent antigen (Ag)-IgE complex triggers mast cell activation, resulting in their degranulation; in the release of inflammatory mediators, proteases, and lipid mediators; and in the production of various cytokines [1]. FcεRI β- and γ-subunits by the Src family non-receptor kinase Lyn. FcεRI β- and γ-subunits by the Src family non-receptor kinase Lyn This is followed by an enhanced activity of tyrosine kinase Syk from the Syk/Zap family and the phosphorylation of transmembrane. An important role in this process is played by protein tyrosine phosphatases [3]

Methods

Results

Conclusion