Chloride Conductance Is Required for the Protein Kinase A and Rac1-dependent Phosphorylation of Moesin at Thr-558 by KCl in PC12 Cells

Songhee Jeon,Sohee Kim,Eunhee Kim,Ji Eun Lee,Sung Joon Kim,Yong-Sung Juhnn,Yong Sik Kim,Chang-Dae Bae,Joobae Park

doi:10.1074/jbc.m408253200

Songhee Jeon, Sohee Kim + Show 7 more

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https://doi.org/10.1074/jbc.m408253200

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Moesin is a member of the ERM family, a family of cross-linkers between the plasma membrane and the actin cytoskeleton, which are known to be activated by phosphorylation. Previously, we reported the RhoA and Rho kinase-dependent phosphorylation of moesin at Thr-558 in hippocampal neuronal cells by glutamate. Here we studied the induction of moesin phosphorylation by KCl (60 mm) in PC12 cells. Moesin phosphorylation at Thr-558 was increased after 2 min of KCl treatment, peaked at 5 min, and returned to the basal level by 60 min. KCl also activated Rac1, but not RhoA, in PC12 cells, and KCl-induced moesin phosphorylation was suppressed in dominant negative Rac1 (N17 Rac1)-expressed cells. The inhibition of protein kinase A (PKA), known as an upstream kinase of Rac1, abolished Rac1 activation and moesin phosphorylation by KCl. Interestingly, the phosphorylation of moesin by KCl was independent of KCl-induced membrane depolarization and calcium influx but was dependent on KCl-induced chloride conductance. 60 mm KCl induced chloride conductance in PC12 cells, and pretreatment with Cl- channel blocker abolished Rac1 activation and moesin phosphorylation by KCl. These results suggest that the phosphorylation of moesin at Thr-558 in PC12 cells by KCl treatment is PKA- and Rac1-dependent and that KCl-induced chloride conductance is involved in the activation of this signaling system.

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Moesin is a member of the ERM1 family proteins, which includes band 4.1 and merlin
To determine whether the RhoA family proteins are involved in the phosphorylation of moesin by KCl, we examined the activities of RhoA, Rac1, and of Cdc42 in PC12 cells after 60 mM KCl treatment by measuring the amounts of RhoA bound to the GST-fused RhoA-binding domain of rhotekin (GST-RBD) or Rac1/Cdc42 bound to the GST-fused Rac/Cdc42-binding domain of PAK3 (GST-PBD)
This study demonstrates that 60 mM KCl induces the phosphorylation of moesin at threonine 558 residue (Thr-558) in PC12 cells protein kinase A (PKA) and Rac1 dependently

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Moesin is a member of the ERM1 (ezrin/radixin/moesin) family proteins, which includes band 4.1 and merlin. 60 mM KCl induced chloride conductance in PC12 cells, and pretreatment with Cl؊ channel blocker abolished Rac1 activation and moesin phosphorylation by KCl. These results suggest that the phosphorylation of moesin at Thr-558 in PC12 cells by KCl treatment is PKA- and Rac1-dependent and that KCl-induced chloride conductance is involved in the activation of this signaling system.

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