K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity.

Jimena Canales,Nicolás Díaz,Elías Leiva-Salcedo,Oscar Cerda,Pablo Cruz,Denise Riquelme

doi:10.3390/cells9102273

Abstract

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.

Highlights

Cell migration is a fundamental process involved in a plethora of physiological and pathophysiological events, such as embryonic development, immune response, wound healing and metastasis of cancer cells [1,2,3,4]
We observed that KCTD5 depletion, obtained through two different shRNA constructs, promoted a two-fold increase in the average of cell migration compared to the respective control cells (Figure 1B)
We evaluated whether Rac1 activity is implicated in the higher cell spreading linked to KCTD5 depletion

Summary

Introduction

Cell migration is a fundamental process involved in a plethora of physiological and pathophysiological events, such as embryonic development, immune response, wound healing and metastasis of cancer cells [1,2,3,4] This process consists of cycles of five consecutive steps: cell polarization, membrane extension at the leading edge, adhesion to cell substratum, generation of traction forces and detachment of rear edge [5]. All those events are the result of the spatial–temporal coordination of several molecular signals, where Rho-GTPases and Ca2+ play a central role [6]. In accordance with the similarity of Ca2+ and Rho GTPases actions in migration-related mechanisms, it has been described that Ca2+ regulates the activity of RhoA and

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Results

Conclusion