Slingshot Phosphatase and LIM Kinase Modulate Mast Cell Activation Beyond the Regulation of Actin Dynamics

Slingshot (SSH) phosphatase and LIM kinase (LIMK) regulate actin dynamics through the dephosphorylation and phosphorylation of cofilin, an actin-severing protein. Antigen stimulation rapidly and temporally induces cofilin dephosphorylation (activation) to depolymerize actin in rat basophilic leukemia (RBL-2H3) cells; however, the roles of SSH and LIMK in mast cell activation remain unclear. Here, we investigated the roles of SSH and LIMK in multivalent antigen-induced mast cell activation. We found that antigen stimulation induces the transient dephosphorylation (activation) of SSH1 and sustained dephosphorylation (inactivation) of LIMK1/2 in RBL-2H3 cells. Pretreatment with the SSH inhibitor D3 prevented cofilin dephosphorylation after antigen stimulation, whereas pretreatment with the LIMK inhibitor BMS-5 suppressed cofilin rephosphorylation, indicating that SSH1 and LIMK1/2 regulate cofilin activity. Pretreatment with D3, but not BMS-5, induced cellular spread and plasma membrane ruffling in resting cells, significantly promoting antigen-induced degranulation. Additionally, SSH1 colocalized with histamine-containing granules, and their colocalization diminished following antigen stimulation. D3 treatment alone prevented this colocalization, suggesting that SSH1 negatively regulates degranulation by modulating secretory granule transportation. Pretreatment with BMS-5 more efficiently suppressed antigen stimulation-induced interleukin-4 secretion when compared to D3. LIMK1 interacted with ERK1/2 in response to antigen stimulation, whereas BMS-5 pretreatment reduced not only the interaction with ERK1/2 but also the phosphorylation and nuclear translocation of ERK1/2. These results suggest that LIMK positively regulates cytokine secretion by promoting ERK1/2 activation. Thus, beyond their role in regulating actin dynamics, SSH and LIMK modulate mast cell activation.