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

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.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome

“…Finally, the microtubule regrowth was determined by capturing the fluorescent signal in both channels from different areas per sample and the sum of fluorescence intensities, representing γ-tubulin and α-tubulin, respectively, was obtained from nine consecutive frames (0.2 μm steps), with the middle frame chosen with respect to the highest γ-tubulin intensity. Intensity quantification of a region of interest, defined as concentric circles of a radius of 1 μm and centered around the γ-tubulin marked centrosome was then done automatically using an in-house written macro for Fiji (51,52).…”

Tubulin: Structure, Functions and Roles in Disease

“…Microtubule nucleation from centrosomes is mediated by γ-tubulin complexes, and it has been suggested that protein tyrosine kinases could modulate microtubule nucleation in activated mast cells [27,28,29]. In their contribution to this Special Issue, Klebanovych and colleagues [30] show that Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6 ) plays an important role in the regulation of microtubules in the later stages of mast cell activation. SHP-1 forms complexes with γ-tubulin complex proteins GCPs and acts as a negative regulator of microtubule nucleation from the centrosomes.…”

C53 interacting with UFM1-protein ligase 1 regulates microtubule nucleation in response to ER stress

“…It has been reported that androgen and Src signaling, which leads to the activation of the ERK, regulate microtubule nucleation by promoting the accumulation of γ-tubulin at the centrosome (Colello et al, 2010). Modulation of γ-tubulin accumulation in centrosomes was also shown for GIT1/βPIX signaling proteins and PAK1 kinase (Černohorská et al, 2016), and for tyrosine phosphatase SHP-1 (Klebanovych et al, 2019).…”

“…We have recently demonstrated that tyrosine-protein phosphatase SHP-1 forms complexes with γTuRC proteins and suppresses microtubule nucleation. This indicates that different protein phosphatases might be involved in distinct signaling pathways with respect to the regulation of microtubule nucleation (Klebanovych et al, 2019). The activation of phosphatase(s) by C53 might maintain a low level of phosphorylated γTuRCs or TuRCtethering proteins, resulting in the attenuation of microtubule nucleation.…”

“…To prepare control C-terminally TagRFP-tagged mouse SHP-1, the coding sequence was excised from pmSHP-1-EGFP plasmid (Klebanovych, 2019) by EcoRI/SalI and ligated into pCI-Tag-RFP (Vinopal et al, 2012), resulting in plasmid pmSHP-1-TagRFP. CRISPR/Cas9 gene editing (Sander and Joung, 2014) was used to disrupt the expression of human UFL1 (Ensembl: ENSG00000014123) or all human CDK5RAP3 variants (Ensembl: ENSG00000108465).…”