Rho kinase in the regulation of cell death and survival

Abstract

Rho kinase (ROCK) belongs to a family of serine/threonine kinases that are activated via interaction with Rho GTPases. ROCK is involved in a wide range of fundamental cellular functions, such as contraction, adhesion, migration, and proliferation. Recent studies have shown that ROCK plays an important role in the regulation of apoptosis in various cell types and animal disease models. Two ROCK isoforms, ROCK1 and ROCK2, are assumed to be function redundant, this based largely on kinase construct overexpression and chemical inhibitors (Y27632 and fasudil) which inhibit both ROCK1 and ROCK2. Gene targeting and RNA interference approaches allow further dissection of distinct cellular, physiological, and patho-physiological functions of the two ROCK isoforms. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of ROCK signaling in the regulation of apoptosis and highlights new findings from recently generated ROCK-deficient mice.

Figure 1. The extrinsic and intrinsic pathways of caspase activation

In the extrinsic pathway, death ligands, such as FasL and TNFα, bind to their respective receptors, leading to caspase 8 activation. Activated caspase 8 directly activates procaspase 3, which then starts death cascade. Caspase 8 can also signal indirectly through the mitochondria via activation of Bid which translocates to mitochondria and activates Bax and Bak to trigger release of cytochrome c (Cyt C). In the intrinsic pathway, many stress stimuli activate caspases through the activation of BH3-only proteins of the Bcl-2 family, such as Bid, Bad, Bim, Nix, etc. The activated BH3-only proteins then activate Bax and Bak, leading to the release of cytochrome c as well as the release of Smac and HtrA2 from mitochondria. This process is antagonized by Bcl-2 and Bcl-X_L. Cytochrome c forms a complex with the adaptor protein Apaf-1, and procaspase 9, resulting in the formation of apoptosome, which leads to the proteolytic cleavage and concomitant activation of caspase 9. Active caspase 9 directly cleaves and activates procaspase 3. This process is antagonized by XIAP, which can be inhibited by Smac and HtrA2 following their release from mitochondria.

Figure 2. Molecular structure of ROCK1 and ROCK2

ROCK sequences comprise a serine/threonine kinase domain located at the amino terminus of the protein, followed by a central coiled-coil domain containing the Rho-binding domain (RBD), and a carboxyl terminal pleckstrin homology (PH) domain with an internal cysteine-rich (CR) domain.

Figure 3. Potential ROCK targets in apoptotic signaling

Several ROCK substrates are involved in the regulation of apoptosis. ROCK can increase MLC phosphorylation through direct effect on MLC or indirectly by inactivating MLC phosphatase (MYPT1). The increased MLC phosphorylation results in stimulation of actomyosin contractility, which regulates morphological apoptotic events during the execution phase of apoptosis including plasma membrane blebbing, nuclear disintegration and fragmentation of apoptotic cells. Under some conditions, the actin cytoskeleton rearrangement induced by ROCK activation can also be involved in the intracellular signaling involved in the initiating stages of apoptosis through the regulation of assembly of death receptor complex or loss of cell adhesion. In addition, ROCK has been shown to stimulate phosphatase and tensin homologue (PTEN) and inhibit insulin receptor substrate 1 (IRS1) signaling (broken line indicates that both positive and negative regulations of IRS1 signaling by ROCK have been reported), resulting in inactivation of Akt. Akt has important roles in promoting cell survival, possibly through inhibition of both extrinsic and intrinsic pathways. Finally, ROCK activation was found to promote apoptosis through increasing ezrin phosphorylation, which in turn led to Fas clustering and membrane expression.