Abstract
BackgroundROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.Methodology/Principal FindingsIn this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.ConclusionsROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.
Highlights
Signalling through Rho family GTPases plays a fundamental role in regulating cell interaction with extracellular matrix (ECM) via heterodimeric adhesion receptors known as integrins [1]
We demonstrated that depletion of ROCK1 and ROCK2 expression has distinct effects on keratinocyte adhesion to fibronectin
Loss of ROCK1 expression resulted in decreased adhesion to fibronectin whereas depletion of ROCK2 resulted in increased adhesion [16]
Summary
Signalling through Rho family GTPases plays a fundamental role in regulating cell interaction with extracellular matrix (ECM) via heterodimeric adhesion receptors known as integrins [1]. Integrins act as bidirectional signal transducers and are clustered into structures generically referred to as adhesion complexes [2]. ROCK1 and ROCK2 share 92% amino acid sequence identity across their kinase domains, sequence identity drops to 65–70% across their PH domains, which may account for the observed differences in cellular localisation of the two isoforms [7,8]. Both isoforms of ROCK play a role in regulating the acto-myosin cytoskeleton through phosphorylation, and inhibition, of the regulatory subunit of myosin light-chain phosphatase [9,10]. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration
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