ROCK1 is a protein serine/threonine kinase also known as rho-associated, coiled-coil-containing protein kinase 1. Other common names are ROKβ and P160ROCK. ROCK1 is a major downstream effector of the small GTPase RhoA and is a regulator of the actomyosin cytoskeleton which promotes contractile force generation. [5] ROCK1 plays a role in cancer and in particular cell motility, metastasis, and angiogenesis. [5]
ROCK1 is also the name of the gene that encodes the protein ROCK1, a serine/threonine kinase. ROCK1 is activated when bound to the GTP-bound form of RhoA. The human ROCK1 gene is located on human chromosome 18 with specific location of 18q11.1. [6] The location of the base pair starts at 18,529,703 and ends at 18,691,812 bp and translates into 1354 amino acids. [7]
ROCK1 has a ubiquitous tissue distribution, but subcellularly it is thought to colocalize with the centrosomes. This is consistent with its function as a key modulator of cell motility, tumor cell invasion, and actin cytoskeleton organization. [7] In rats, ROCK1 is expressed in the lung, liver, spleen, kidney, and testis. [8] [9] [10]
The ROCK1 structure is a serine/threonine kinase with molecular weight of 158 kDa. [7] It is a homodimer composed of a catalytic kinase domain (residues76-338) [11] located at the amino or N-terminus of the protein, a coiled-coil region (residues 425-1100) [11] containing the Rho-binding domain, and a pleckstrin-homology domain (residues 1118-1317) [11] with a cysteine-rich domain. When a substrate is absent, ROCK1 is an autoinhibited loop structure. Enzyme activity of ROCK1 is inhibited when the pleckstrin-homology and Rho-binding domains in the C-terminus independently bind to the N-terminus kinase domain. When a substrate such as GTP-bound RhoA binds to the Rho-binding region of the coiled-coil domain, the interactions between the N-terminus and the C-terminus are disrupted, thus activating the protein. Cleavage of the C-terminal inhibitory domain by caspase-3 during apoptosis can also activate the kinase. [12]
This view of autoinhibition released by RhoA binding has been challenged by low resolution electron microscopy data showing ROCK to be a constitutive linear dimer 120 nm in length. [13] According to this new data ROCK does not need to be activated by RhoA or phosphorylation because it is always active, and whether ROCK will phosphorylate its substrates (e.g. myosin regulatory light chain) depends only on their subcellular localization. [13]
There is one other isoform of ROCK known as ROCK2. ROCK2 is located at 2p24 and is highly homologous with ROCK1 with an overall amino acid sequence identity of 65%. [11] The identity in the Rho-binding domain is 58% [11] and approximately 92% [11] in the kinase domain. The ROCK isoforms are encoded by two different identified genes and are ubiquitously expressed. [11]
GTPase-RhoA binding can increase the activity of ROCK1 by 1.5-2-fold. [14] Without RhoA binding, lipids such as arachidonic acid or sphingosine phosphorylcholine can increase ROCK1 activity 5- to 6-fold. [14] [15] These two lipids interact with the pleckstrin-homology domain, thus disrupting its ability to inhibit ROCK1. [16] G-protein RhoE binds to the N-terminus of ROCK1 and inhibits its activity by preventing RhoA binding. Small G-proteins, Gem and Rad, have been shown to bind and inhibit ROCK1 function, but their mechanism of action is unclear. [11]
ROCK1 phosphorylation sites are at RXXS/T or RXS/T. [11] More than 15 ROCK1 substrates have been identified and activation from these substrates most often leads to actin filament formation and cytoskeleton rearrangements. [11] MYPT-1 is involved in a pathway for smooth muscle contraction. When ROCK1 is activated by binding of GTPase RhoA it produces multiple signaling cascades. For example, RhoA is one of the downstream signaling cascades activated by vascular endothelial growth factor (VEGF). ROCK1 acts as a negative regulator of VEGF endothelial cell activation and angiogenesis. [17] ROCK1 activation by RhoA also promotes stabilization of F-actin, phosphorylation of regulatory myosin light chain (MLC) and an increase in contractility, which plays a crucial role in tumor cell migration and metastasis. [18] This activated ROCK1 also activates LIM kinase, which, phosphorylates cofilin, inhibiting its actin-depolymerizing activity. [19] This depolymerization results in stabilization of actin filaments and decreased branching which promotes contraction.
Cardiac troponin is another ROCK1 substrate that upon phosphorylation causes reduction in tension in cardiac myocytes. [11] ROCK1 also acts as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability.
ROCK1 has a diverse range of functions in the body. It is a key regulator of actin-myosin contraction, stability, and cell polarity. [17] These contribute to many progresses such as regulation of morphology, gene transcription, proliferation, differentiation, apoptosis and oncogenic transformation. [5] Other functions involve smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility. These functions are activated by phosphorylation of DAPK3, GFAP, LIMK1, LIMK2, MYL9/MLC2, PFN1 and PPP1R12A. [17] Additionally, ROCK1 phosphorylates FHOD1 and acts synergistically with it to promote SRC-dependent non-apoptotic plasma membrane blebbing. [17] It is also required for centrosome positioning and centrosome-dependent exit from mitosis. [17]
ROCK1 has been shown to interact with:
In humans, the main function of ROCK1 is actomyosin contractility. As mentioned before, this contributes to many proximal progresses such as regulation of morphology, motility, and cell–cell and cell–matrix adhesion. [5] In addition, ROCK kinases influence more distal cellular processes including gene transcription, proliferation, differentiation, apoptosis and oncogenic transformation. [5] Given this diverse range of functions, it is not surprising that ROCK1 has been implicated in numerous aspects of cancer. [5]
Recent studies have explored the role of ROCK1 in cancer with particular attention focused on cell motility, metastasis, and angiogenesis. [5] Rho GTPases such as RhoA are highly involved in morphologic changes in cells. When a tumor progresses from invasive to metastatic form it requires that they undergo these dramatic morphologic changes. Therefore, increased expression of RhoA and its downstream effector ROCK1 is often observed in human cancers. These cancers are typically more invasive and metastatic phenotypes. [24]
Increased expression of RhoA and ROCK1 in endothelial cell migration pathways can cause an increase in angiogenesis and metastatic behavior in tumor cells. [24] It has been suggested that ROCK1 either regulates the expression of angiogenic factors or ROCK1 activation facilitates angiogenesis by increasing the plasticity of the tumor. By reducing the strength of cell-cell interactions and aiding the movement of tumor cells, ROCK1 may enable endothelial cells to penetrate the tumor mass more easily. [24]
ROCK1 inhibitors might be used in cancer therapy for:
ROCK1 inhibition for cancer treatment has not been approved for standard therapy use. Y27632 and Fasudil are examples of ROCK1 inhibitors. Both inhibit ROCK1 by competing with ATP for the kinase activation site. Experiments with Y27632 show it is a promising candidate as a therapeutic antihypertensive agent. [11] Fasudil has been used to characterize the role of ROCK1 in vascular function in clinical studies and has been approved for use in Japan for treatment of cerebral vasospasm following subarachnoid hemorrhage. [11]
The ROCK1 signaling plays an important role in many diseases including diabetes, neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), [25] and pulmonary hypertension. [26]
Angiomotin (AMOT) is a protein that in humans is encoded by the AMOT gene. It belongs to the motin family of angiostatin binding proteins, which includes angiomotin, angiomotin-like 1 (AMOTL1) and angiomotin-like 2 (AMOTL2) characterized by coiled-coil domains at N-terminus and consensus PDZ-binding domain at the C-terminus. Angiomotin is expressed predominantly in endothelial cells of capillaries as well as angiogenic tissues such as placenta and solid tumor.
The Rho family of GTPases is a family of small signaling G proteins, and is a subfamily of the Ras superfamily. The members of the Rho GTPase family have been shown to regulate many aspects of intracellular actin dynamics, and are found in all eukaryotic kingdoms, including yeasts and some plants. Three members of the family have been studied in detail: Cdc42, Rac1, and RhoA. All G proteins are "molecular switches", and Rho proteins play a role in organelle development, cytoskeletal dynamics, cell movement, and other common cellular functions.
Cell division control protein 42 homolog is a protein that in humans is encoded by the CDC42 gene. Cdc42 is involved in regulation of the cell cycle. It was originally identified in S. cerevisiae (yeast) as a mediator of cell division, and is now known to influence a variety of signaling events and cellular processes in a variety of organisms from yeast to mammals.
Ras-related C3 botulinum toxin substrate 1, is a protein that in humans is encoded by the RAC1 gene. This gene can produce a variety of alternatively spliced versions of the Rac1 protein, which appear to carry out different functions.
Transforming protein RhoA, also known as Ras homolog family member A (RhoA), is a small GTPase protein in the Rho family of GTPases that in humans is encoded by the RHOA gene. While the effects of RhoA activity are not all well known, it is primarily associated with cytoskeleton regulation, mostly actin stress fibers formation and actomyosin contractility. It acts upon several effectors. Among them, ROCK1 and DIAPH1 are the best described. RhoA, and the other Rho GTPases, are part of a larger family of related proteins known as the Ras superfamily, a family of proteins involved in the regulation and timing of cell division. RhoA is one of the oldest Rho GTPases, with homologues present in the genomes since 1.5 billion years. As a consequence, RhoA is somehow involved in many cellular processes which emerged throughout evolution. RhoA specifically is regarded as a prominent regulatory factor in other functions such as the regulation of cytoskeletal dynamics, transcription, cell cycle progression and cell transformation.
PTK2 protein tyrosine kinase 2 (PTK2), also known as focal adhesion kinase (FAK), is a protein that, in humans, is encoded by the PTK2 gene. PTK2 is a focal adhesion-associated protein kinase involved in cellular adhesion and spreading processes. It has been shown that when FAK was blocked, breast cancer cells became less metastatic due to decreased mobility.
Serine/threonine-protein kinase PAK 1 is an enzyme that in humans is encoded by the PAK1 gene.
LIM domain kinase 1 is an enzyme that in humans is encoded by the LIMK1 gene.
Ras GTPase-activating-like protein IQGAP1 (IQGAP1) also known as p195 is a ubiquitously expressed protein that in humans is encoded by the IQGAP1 gene. IQGAP1 is a scaffold protein involved in regulating various cellular processes ranging from organization of the actin cytoskeleton, transcription, and cellular adhesion to regulating the cell cycle.
RhoC is a small signaling G protein, and is a member of the Rac subfamily of the family Rho family of GTPases. It is encoded by the gene RHOC.
Serine/threonine-protein kinase PAK 4 is an enzyme that in humans is encoded by the PAK4 gene.
Deleted in Liver Cancer 1 also known as DLC1 and StAR-related lipid transfer protein 12 (STARD12) is a protein which in humans is encoded by the DLC1 gene.
Rnd3 is a small signaling G protein, and is a member of the Rnd subgroup of the Rho family of GTPases. It is encoded by the gene RND3.
Cyclin-dependent kinase 5 is a protein, and more specifically an enzyme, that is encoded by the Cdk5 gene. It was discovered 15 years ago, and it is saliently expressed in post-mitotic central nervous system neurons (CNS).
The Akt signaling pathway or PI3K-Akt signaling pathway is a signal transduction pathway that promotes survival and growth in response to extracellular signals. Key proteins involved are PI3K and Akt.
Dishevelled (Dsh) is a family of proteins involved in canonical and non-canonical Wnt signalling pathways. Dsh is a cytoplasmic phosphoprotein that acts directly downstream of frizzled receptors. It takes its name from its initial discovery in flies, where a mutation in the dishevelled gene was observed to cause improper orientation of body and wing hairs. There are vertebrate homologs in zebrafish, Xenopus (Xdsh), mice and humans. Dsh relays complex Wnt signals in tissues and cells, in normal and abnormal contexts. It is thought to interact with the SPATS1 protein when regulating the Wnt Signalling pathway.
Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC family of serine-threonine specific protein kinases. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton.
In molecular biology, the IMD domain is a BAR-like domain of approximately 250 amino acids found at the N-terminus in the insulin receptor tyrosine kinase substrate p53 (IRSp53/BAIAP2) and in the evolutionarily related IRSp53/MIM (MTSS1) family. In IRSp53, a ubiquitous regulator of the actin cytoskeleton, the IMD domain acts as conserved F-actin bundling domain involved in filopodium formation. Filopodium-inducing IMD activity is regulated by Cdc42 and Rac1 and is SH3-independent. The IRSp53/MIM family is a novel F-actin bundling protein family that includes invertebrate relatives:
XB130 is a cytosolic adaptor protein and signal transduction mediator. XB130 regulates cell proliferation, cell survival, cell motility and gene expression. XB130 is highly similar to AFAP and is thus known as actin filament associated protein 1-like 2 (AFAP1L2). XB130 is a substrate and regulator of multiple tyrosine kinase-mediated signaling. XB130 is highly expressed in the thyroid and spleen.
Rho-kinase inhibitors are a series of compounds that target rho kinase (ROCK) and inhibit the ROCK pathway. Clinical trials have found that inhibition of the ROCK pathway contributes to the cardiovascular benefits of statin therapy. Furthermore, ROCK inhibitors may have clinical applications for anti-erectile dysfunction, antihypertension, and tumor metastasis inhibition. More recently they have been studied for the treatment of glaucoma and as a therapeutic target for the treatment of cardiovascular diseases, including ischemic stroke. While statin therapy has been demonstrated to reduce the risk of major cardiovascular events, including ischemic stroke, the interplay between the ROCK pathway and statin therapy to treat and prevent strokes in older adults has not yet been proven.
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