Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

Jianjian Shi,Yu Yang,Reuben Kapur,Sasidhar Vemula,Lei Wei,Lumin Zhang,Xiangbing Wu,Michelle Surma

doi:10.1038/cddis.2013.10

Jianjian Shi, Yu Yang + Show 6 more

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https://doi.org/10.1038/cddis.2013.10

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Abstract

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1−/− and ROCK2−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

Highlights

The ROCK family contains two members, ROCK1 and ROCK2; they share 65% overall identity and 92% identity in the kinase domain.[1,2,3,4] Both kinases contain a catalytic kinase domain at the N-terminus, followed by a central coiled-coil domain, including a Rho-binding domain and a C-terminal pleckstrin-homology (PH) domain, with an internal cysteine-rich domain
Using mouse embryonic fibroblast (MEF) cells derived from ROCK1 knockout mice,[22] the current study reveals a novel role for ROCK1 in mediating actin cytoskeleton remodeling in response to cytotoxic stress induced by doxorubicin, a chemotherapeutic drug.[23,24]
The present study reveals the distinct roles of ROCK isoforms in regulating actin cytoskeleton reorganization and cell detachment induced by doxorubicin

Summary

Introduction

The ROCK family contains two members, ROCK1 and ROCK2; they share 65% overall identity and 92% identity in the kinase domain.[1,2,3,4] Both kinases contain a catalytic kinase domain at the N-terminus, followed by a central coiled-coil domain, including a Rho-binding domain and a C-terminal pleckstrin-homology (PH) domain, with an internal cysteine-rich domain. DNA, inhibit DNA topoisomerase II, and generate free radicals.[25,26] At the same time, doxorubicin increases actin cytoskeleton instability via inhibition of actin polymerization.[27,28] We observed that ROCK1 deficiency in MEF cells inhibited actin cytoskeleton reorganization by attenuating periphery actomyosin ring formation and preserving central stress fibers, resulting in decreased cell detachment and predetachment apoptosis. These protective features are unique to ROCK1 deficiency as ROCK2-deficient cells exhibited increased periphery membrane folding and altered cell adhesion. Our results reveal that ROCK1 and ROCK2 are functionally different in regulating stress-induced actin cytoskeleton reorganization and cell detachment

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Conclusion