Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_328


Historical Background

The Rho family of small molecular weight GTP-binding proteins, which comprises 22 proteins including RhoA, Rac1, and Cdc42, was originally isolated based on their high degree of homology with the Ras proto-oncogenes (Rho = Ras homologue). Following their identification, a variety of approaches were used to isolate interacting proteins that might convey signals downstream to regulate numerous biological processes. One approach was to use GTP-loaded RhoA as a high-affinity reagent to fish for interacting proteins, and the ROCK kinases were identified in this way (Leung et al. 1995; Ishizaki et al. 1996; Matsui et al. 1996). A number of different names have been used to describe the two kinases, and early reports were sometimes not careful to accurately specify which isoforms were actually used in the studies. However, the official gene names have been set as ROCK1 and ROCK2. The function of ROCK kinases downstream of active RhoA was quickly determined to be the regulation of actin–myosin contractile force generation (Fig. 1), mediated via the phosphorylation of a number of substrates including LIMK (which in turn phosphorylates cofilin and inactivates its actin-severing function), myosin light chain (MLC), and the myosin-binding subunit of the MLC phosphatase (MYPT1) (Julian and Olson 2014). In cultured cells, ROCK activation leads to the formation of actin stress fibers and focal adhesions. The discovery of the selective ROCK inhibitor Y27632 (Uehata et al. 1997) rapidly advanced the understanding of ROCK contributions to numerous biological functions.
ROCK, Fig. 1

ROCK pathways leading to increased actin-myosin contractility. Active GTP-bound Rho associates with ROCK and increases specific activity with the consequence of increased phosphorylation of proteins including the MYPT1 myosin-binding subunit of the myosin light chain phosphatase. ROCK phosphorylation of MYPT1 affects both its substrate binding and catalytic activity, resulting in inhibition of myosin light chain dephosphorylation. MLC has also been reported to be directly phosphorylated and activated by ROCK. ROCK phosphorylation of LIMK1 and LIMK2 increases their specific activity, resulting in phosphorylation and inactivation of cofilin family proteins. Cofilin phosphorylation inhibits its filamentous actin-severing activity. The sum total of these events is stabilization of filamentous actin and increased actin-myosin contractility

Structure and Function of ROCK Kinases

The ROCK kinases are both 160 kDa serine–threonine kinases with identical domain organization. The primary sequence of the kinase domains places ROCK in the AGC family, their closest relatives being the myotonic dystrophy kinase (DMPK) and the myotonic dystrophy kinase-related CDC42-binding kinases (MRCK) (Unbekandt and Olson 2014). As depicted in Fig. 2, the ROCK kinases are composed of an N-terminal kinase domain, a coiled-coil region that contains the Rho-binding domain (RBD), and a pleckstrin homology (PH) domain that is split into two portions on either side of a cysteine-rich C1 domain. The high degree of homology between the ROCK1 and ROCK2 kinase domains suggests that they probably phosphorylate the same substrates and experimental data bears out this conclusion. Differences in substrate phosphorylation or biological functions observed in vivo likely result from different subcellular localization and/or protein–protein interactions. Crystal structures of the kinase domains alone or liganded with various small-molecule inhibitors have been reported that have revealed some novel aspects of the kinase domain. In comparison to other related kinases, there are extensions to both N-terminal and C-terminal ends that promote dimerization. Also, unlike most other AGC kinases, phosphorylation in the kinase activation loop or hydrophobic motifs does not appear to be required for ROCK activation. Instead, dimerization appears to allow the kinase to adopt an active conformation without the need for phosphorylation. NMR structural information for the PH and C1 domains of ROCK2 has also been reported (Wen et al. 2008). Activity of the kinase domain is autoinhibited by the C-terminus, which can be relieved through binding to RhoA, RhoB, or RhoC (Leung et al. 1995; Ishizaki et al. 1996; Matsui et al. 1996) or through proteolytic cleavage (Coleman et al. 2001; Sebbagh et al. 2005). Multiple sites in the C-terminus appear to interact with the kinase domain to regulate activity (Lochhead et al. 2010). Interestingly, a single proline to serine amino acid substitution within the first portion of the split PH domain was sufficient to activate ROCK1, suggesting that the conformation of the C-terminal region is important for kinase regulation (Lochhead et al. 2010). In the case of ROCK2, electron microscopy revealed that the dimerized protein contains globular kinase and C-terminal regions separated by a semirigid extended coiled coil (Truebestein et al. 2015, 2016). These results suggested that the C-terminal regions orient towards the face of the inner plasma membrane, with the coiled-coil region keeping the dimeric kinase domains spaced at a fixed distance away from the membrane where substrates would be phosphorylated. ROCK1 activity may be inhibited by another Rho family member RhoE (Riento et al. 2003), which in turn can be antagonized by the kinase PDK1 in a manner independent of its catalytic activity (Pinner and Sahai 2008). Given the key role that ROCK kinases play in promoting contractile force generation, it is not surprising that they have been found to make important contributions to fundamental processes including motility, adhesion, cytokinesis, apoptosis, phagocytosis, smooth muscle contraction, and neurite retraction.
ROCK, Fig. 2

ROCK functional domains. Common functional domains in human ROCK1 and ROCK1 with the positions of starting and ending residues. The percentage identities between matched regions were determined by pairwise BLAST comparisons. RBD Rho binding domain, PH pleckstrin homology domain, C1 protein kinase C conserved region 1 (Not to scale)

ROCK in Development

Genetic deletion of either ROCK1 (Shimizu et al. 2005) or ROCK2 (Thumkeo et al. 2003) in mice resulted in similar phenotypes, which helped reveal that a major function of the ROCK kinases in vivo is the regulation of epithelial cell motility. The homozygous deletion of ROCK1 still allowed for the birth of mice at the expected Mendelian ratios, indicating that there were no major problems during growth and development in utero, but newborns had defects in eyelid and ventral body wall closure that gave rise to eyes-open at birth (EOB) and omphalocele (organs such as the liver and gut not being contained with the abdomen) phenotypes, respectively. EOB and omphalocele were also observed in homozygous ROCK2 knockout mice, but in this case a sub-Mendelian incidence of ROCK2-/- mice resulted from defects in the placental labyrinth layer, causing decreased blood flow to developing ROCK2-/- embryos (Thumkeo et al. 2003, 2005). Given these results, it seems consistent that ROCK1+/-; ROCK2+/- double heterozygous mice also exhibited EOB and omphalocele, indicating that both kinases contribute to the same actin-driven movement and reorganization of epithelial sheets for eyelid and ventral body wall closure (Thumkeo et al. 2005). That being said, homozygous ROCK1 knockout mice were also independently generated, showing no obvious phenotypic differences from wild-type littermates, which suggests that strain background differences may contribute to the penetrance of the ROCK1 deficient phenotype. (Zhang et al. 2006).

ROCK in Disease

The ready availability of potent and selective ROCK inhibitors has made it possible to examine whether ROCK kinases are involved in a wide variety of pathological conditions including cancer, hypertension and cardiovascular disease, neuronal degeneration, kidney failure, clotting diseases, asthma, glaucoma, osteoporosis, erectile dysfunction, and insulin resistance. However, the areas that have attracted the most research effort are cancer, hypertension–cardiovascular disease, and glaucoma.

Interest in ROCK as a cancer target stems from its wide range of activities that contribute to the growth and progression of tumors including proliferation, survival, and metastasis (reviewed in Wickman et al. (2010)). Early studies revealed that Rho GTPs are overexpressed in a variety of tumors, consistent with increased signaling through the ROCK pathway being a contributory factor. In addition, elevated ROCK expression has been reported in bladder (Kamai et al. 2003) and testicular cancers (Kamai et al. 2002) and correlates with poor survival (Kamai et al. 2003). Conditional deletion of both ROCK1 and ROCK2 in mouse models of lung cancer and melanoma revealed that loss of both kinases blocked tumor formation, indicating that they have essential and redundant roles in tumorigenesis (Kümper et al. 2016). Large-scale sequencing efforts directed at the identification of genetic alterations in human cancers revealed a number of activating somatic ROCK1 mutations in human tumors and tumor cell lines (Lochhead et al. 2010). Accordingly, conditional activation of a ROCK transgene within the epidermis strongly promoted tumor progression in a model of cutaneous squamous cell carcinoma, while pharmacological inhibition of ROCK activity suppressed it (Samuel et al. 2011). A current concept is that it would be advantageous to develop ROCK2 selective inhibitors for the treatment of cancer to avoid the pronounced hypotension that is a side effect of ROCK1 inhibition.

The connection between ROCK and hypertension was revealed with the development of ROCK selective inhibitors. Inhibition of ROCK with Y27632 and related compounds was shown to relieve hypertension in rats by inhibiting the calcium sensitization of smooth muscle contraction. Since that time, numerous studies have built on these observations to show that ROCK activity mediates increased smooth muscle contraction principally via modulation of MLC phosphorylation. In particular, ROCK appears to contribute to aberrant vascular contraction, for example, during coronary vasospasm, cerebral vasospasm following subarachnoid hemorrhage, pulmonary hypertension, and Raynaud’s phenomenon, a condition in which the blood supply to distal extremities such as fingers and toes is decreased to the point of numbness or pain and which is the result of vasospasms. Consistent with a role for ROCK in the regulation of vascular contraction and blood pressure, ubiquitous expression of a conditionally activated ROCK2 transgene resulted in cerebral hemorrhagic lesions (Samuel et al. 2016).

Glaucoma is a disease in which damage to the optic nerve progressively leads to impaired vision and possibly blindness. One way that optic nerve damage may occur is through elevated intraocular pressure. ROCK inhibition helps to relieve this pressure by increasing aqueous outflow by reducing MLC phosphorylation in cells lining the trabecular meshwork. Significant research by pharmaceutical companies in this area has resulted in several promising clinical trials.

Within the nervous system, ROCK has been shown to be an important trigger of neuronal growth cone collapse and neurite withdrawal. As a result, there has been considerable interest in the possibility that ROCK inhibition would actually promote neurite outgrowth and dendrite formation. Possible applications for this include recovery from spinal cord injury by assisting the reestablishment of neural connections across the lesion and Alzheimer’s disease treatment through the decreased production of amyloid precursor protein.

ROCK and Stem Cell Survival

When human embryonic stem cells (hESC) are dissociated or plated at low density, they often undergo apoptotic death. Chemical biology screens to identify agents that would promote survival identified the ROCK selective inhibitor Y27632 as a particularly potent agent. Mechanistic studies revealed that ROCK-mediated actin–myosin contraction makes epiblast-derived hESCs die during low density growth and validate the use of inhibitors of ROCK or actin–myosin contractility for the propagation and genetic manipulation of hESCs for eventual therapeutic use (Samuel and Olson 2010).

ROCK Inhibitors

  1. 1.

    HA-1077 (fasudil) and hydroxyfasudil have been in clinical use in Japan for cerebral vasospasm since 1995 (Olson 2008). Since it has been used for such a long period, there is positive post-marketing safety data, which has encouraged trials for a number of indications including angina, acute ischemic stroke, cerebral blood flow, stable angina pectoris, coronary artery spasm, heart failure-associated vascular resistance and constriction, pulmonary arterial hypertension, essential hypertension, atherosclerosis, and aortic stiffness (Olson 2008).

  2. 2.

    Y-27632 was the first published selective ROCK inhibitor (Uehata et al. 1997), and its ready availability has made it the inhibitor of choice. Although this inhibitor is not strictly specific for ROCK kinases, ROCK1 and ROCK2 siRNA experiments have not revealed significant off-target effects in cells.

  3. 3.

    H-1152 was developed as an improved version of HA-1077 with greater ROCK selectivity over PKA and PKC. Although also readily available from commercial sources, H-1152 is less often used than HA-1077 to corroborate results from experiments in which Y-27632 was used, despite the improved selectivity.

  4. 4.

    KD025 (formerly known as SLx-2119) was developed as a potential cancer therapeutic initially but more recently has gone into clinical development for the treatment of diseases including psoriasis vulgaris, chronic graft-versus-host disease, and pulmonary fibrosis (Zanin-Zhorov et al. 2016).

  5. 5.

    AT13148 was originally developed as an AKT inhibitor but was found to be a broad specificity inhibitor for several related AGC family kinases. Recent research has shown that the high potency of AT13148 for ROCK1 and ROCK2 is chiefly responsible for the antitumor properties of this compound using in vivo models (Yap et al. 2012). AT13148 has entered Phase I clinical trials in patients with advanced solid tumors.

A number of studies have highlighted the fact that no inhibitor is entirely specific. Therefore, greater robustness can be built into studies that make use of ROCK inhibitors if a number of additional conditions were satisfied including:
  1. 1.

    Structurally unrelated inhibitors should produce the same biological effects at concentrations that produce equivalent kinase inhibition. The lowest effective doses should be used to reduce off-target effects.

  2. 2.

    Where possible additional methods should be used to inhibit ROCK function, such as RNAi-mediated knockdown or CRISPR–Cas9-mediated gene disruption, and the biological effects of inhibitors should be consistent with those observed using these alternative approaches.

  3. 3.

    Dose–response experiments to establish rank order of potency for a set of inhibitors, i.e., the most potent ROCK inhibitors should be the most effective if a biological response is mediated by ROCK.

  4. 4.

    Examination of the relationship between ROCK inhibitor dose, substrate phosphorylation, and biological endpoint.


Although there is no doubt that inhibitors are useful and convenient research tools, care should be taken in interpreting the results. The substantial knowledge base of the biological functions of ROCK has been made possible due to the ready availability of such inhibitors. Their greatest utility is actually in excluding a possible involvement of ROCK in specific biological responses when there are adequate positive controls in place.


The most important and central cellular function of ROCK kinases is to regulate cell morphology, largely through actin–myosin contractility. Due to its profound influence on morphology and contractility, ROCK directly influences numerous activities, such as cytokinesis, adhesion, motility, endothelial barrier function, and membrane blebbing. In addition, via direct or indirect pathways, the ROCK kinases also influence biological processes including gene transcription, proliferation, regulation of cell size, and survival. There has been considerable interest in ROCK kinases as potential therapeutic targets for cancer, hypertension–cardiovascular disease and glaucoma, and a number of potent and selective inhibitors have been discovered. In fact, one ROCK inhibitor fasudil has been used clinically in Japan for a number of years for the treatment of cerebral vasospasm. Although there is a substantial literature on ROCK function, largely generated due to the availability of pharmacological inhibitors, greater knowledge at the tissue and organismal levels will result from conditional knockout (Kümper et al. 2016) and conditional-activation mouse models (Samuel et al. 2016, 2009). In vivo experiments using these types of genetically modified models will validate the role of ROCK in various pathological conditions and will highlight additional indications for the use of ROCK inhibitors. Although quite a few ROCK substrates have been identified and well characterized, recent phosphoproteomic studies have identified a large number of previously unknown ROCK substrates. As a result, a significant opportunity awaits to characterize the biological outcomes of ROCK-mediated phosphorylation on these novel substrates and ultimately to determine their possible contributions to human disease.


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Centre for Cancer Biology, SA Pathology and University of South AustraliaAdelaideAustralia