Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC (PKA/ PKG/PKC) family of serine-threonine specific protein kinases. It is involved mainly in regulating the shape and movement of cells by acting on the

cytoskeleton The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is com ...

. ROCKs (

ROCK1 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 effecter of the small GTPase RhoA and is a regulator of the a ...

and ROCK2) occur in mammals (human, rat, mouse, cow), zebrafish, ''

Xenopus ''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-know ...

'', invertebrates ('' C. elegans'', mosquito, ''

Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many speci ...

'') and chicken. Human ROCK1 has a

molecular mass The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The related quanti ...

of 158

kDa The dalton or unified atomic mass unit (symbols: Da or u) is a non-SI unit of mass widely used in physics and chemistry. It is defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at re ...

and is a major downstream effector of the small

GTPase GTPases are a large family of hydrolase enzymes that bind to the nucleotide guanosine triphosphate (GTP) and hydrolyze it to guanosine diphosphate (GDP). The GTP binding and hydrolysis takes place in the highly conserved P-loop "G domain", a pro ...

RhoA 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 ...

. Mammalian ROCK consists of a kinase domain, a coiled-coil region and a

Pleckstrin homology Pleckstrin homology domain (PH domain) or (PHIP) is a protein domain of approximately 120 amino acids that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton. This domain can bind phospha ...

(PH) domain, which reduces the kinase activity of ROCKs by an autoinhibitory intramolecular fold if RhoA-GTP is not present. Rat ROCKs were discovered as the first effectors of Rho and they induce the formation of

stress fiber Stress fibers are contractile actin bundles found in non-muscle cells. They are composed of actin (microfilaments) and non-muscle myosin II (NMMII), and also contain various crosslinking proteins, such as α-actinin, to form a highly regulated ...

s and

focal adhesions In cell biology, focal adhesions (also cell–matrix adhesions or FAs) are large macromolecular assemblies through which mechanical force and regulatory signals are transmitted between the extracellular matrix (ECM) and an interacting cell. More ...

by phosphorylating MLC (myosin light chain). Due to this phosphorylation, the

actin Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ov ...

binding of

myosin II Myosins () are a superfamily of motor proteins best known for their roles in muscle contraction and in a wide range of other motility processes in eukaryotes. They are ATP-dependent and responsible for actin-based motility. The first myosin (M2 ...

and, thus, the

contractility Contractility refers to the ability for self-contraction, especially of the muscles or similar active biological tissue *Contractile ring in cytokinesis *Contractile vacuole *Muscle contraction **Myocardial contractility *See contractile cell for ...

increases. Two mouse ROCK isoforms

and ROCK2 have been identified. ROCK1 is mainly expressed in the lung,

liver The liver is a major organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the synthesis of proteins and biochemicals necessary for digestion and growth. In humans, it ...

spleen The spleen is an organ found in almost all vertebrates. Similar in structure to a large lymph node, it acts primarily as a blood filter. The word spleen comes .

kidney The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blo ...

and

testis A testicle or testis (plural testes) is the male reproductive gland or gonad in all bilaterians, including humans. It is homologous to the female ovary. The functions of the testes are to produce both sperm and androgens, primarily testostero ...

. However, ROCK2 is distributed mostly in the

brain A brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. It is the most complex organ in a ve ...

and

heart The heart is a muscular organ in most animals. This organ pumps blood through the blood vessels of the circulatory system. The pumped blood carries oxygen and nutrients to the body, while carrying metabolic waste such as carbon dioxide to t ...

. Protein kinase C and Rho-associated protein kinase are involved in regulating calcium ion intake; these calcium ions, in turn stimulate a myosin light chain kinase, forcing a contraction. Rho-associated protein kinase are serine or threonine kinases that determine the calcium sensitivity in smooth muscle cells.

Function

ROCK plays a role in a wide range of different cellular phenomena, as ROCK is a downstream effector protein of the small

Rho, which is one of the major regulators of the

. 1. ROCK is a key regulator of actin organization and thus a regulator of

cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular dire ...

as follows: Different substrates can be phosphorylated by ROCKs, including LIM kinase, myosin light chain (MLC) and MLC phosphatase. These substrates, once phosphorylated, regulate actin filament organization and contractility as follows: *Amount of actin filaments ROCK inhibits the depolymerization of actin filaments indirectly: ROCK phosphorylates and activates

LIM kinase LIM kinase-1 (LIMK1) and LIM kinase-2 (LIMK2) are actin-binding kinases that phosphorylate members of the ADF/cofilin family of actin binding and filament severing proteins. ADF/cofilin are the only substrates yet identified for the LIM kinases. ...

, which in turn phosphorylates ADF/cofilin, thereby inactivating its actin-depolymerization activity. This results in the stabilization of actin filaments and an increase in their numbers. Thus, over time actin monomers that are needed to continue actin polymerization for migration become limited. The increased stable actin filaments and the loss of actin monomers contribute to a reduction of cell migration. *Cellular contractility ROCK also regulates cell migration by promoting cellular contraction and thus cell-substratum contacts. ROCK increases the activity of the motor protein myosin II by two different mechanisms: :*Firstly, phosphorylation of the myosin light chain ( MLC) increases the myosin II ATPase activity. Thus several bundled and active myosins, which are asynchronously active on several actin filaments, move actin filaments against each other, resulting in the net shortenting of actin fibres. :*Secondly, ROCK inactivates MLC phosphatase, leading to increased levels of phosphorylated MLC. Thus in both cases, ROCK activation by Rho induces the formation of actin

s, actin filament bundles of opposing polarity, containing myosin II, tropomyosin, caldesmon and MLC-kinase, and consequently of focal contacts, which are immature

integrin Integrins are transmembrane receptors that facilitate cell-cell and cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cellular signals such as regulation of the cell cycle ...

-based adhesion points with the extracellular substrate. 2. Other functions and targets *RhoA-GTP stimulates the phospholipid phosphatase activity of PTEN ( phosphatase and tensin homologue), a human

tumor A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...

suppressor protein. This stimulation seems to depend on ROCK. In this way, PTEN is important to prevent uncontrolled cell division as is exhibited in cancer cells. *ROCK plays an important role in cell cycle control, it seems to inhibit the premature separation of the two

centrioles In cell biology a centriole is a cylindrical organelle composed mainly of a protein called tubulin. Centrioles are found in most eukaryotic cells, but are not present in conifers (Pinophyta), flowering plants (angiosperms) and most fungi, and are ...

in G1, and is proposed to be required for contraction of the cleavage furrow, which is necessary for the completion of

cytokinesis Cytokinesis () is the part of the cell division process during which the cytoplasm of a single eukaryotic cell divides into two daughter cells. Cytoplasmic division begins during or after the late stages of nuclear division in mitosis and mei ...

. *ROCKs also seem to antagonize the insulin signaling pathway, resulting in a reduction of cell size and influence cell fate. *ROCKS play a role in membrane blebbing, a morphological change seen in cells committed to apoptosis. The pro-apoptotic protease, caspase 3, activates ROCK kinase activity by cleaving the C-terminal PH domain. As a result, the autoinhibitory intramolecular fold of ROCK is abolished. ROCK also regulates MLC phosphorylation and actomyosin contractility, which regulate membrane blebbing. *ROCKs contribute to

neurite A neurite or neuronal process refers to any projection from the cell body of a neuron. This projection can be either an axon or a dendrite. The term is frequently used when speaking of immature or developing neurons, especially of cells in cultur ...

retraction by inducing

growth cone A growth cone is a large actin-supported extension of a developing or regenerating neurite seeking its synaptic target. It is the growth cone that drives axon growth. Their existence was originally proposed by Spanish histologist Santiago Ram� ...

collapse by activating actomyosin contractility. It is also possible that phosphorylation of collapsin response mediator protein-2 (CRMP2) by ROCK inhibits CRPM2 function of promoting axon outgrowth, resulting in growth cone collapse. *ROCKs regulate cell-cell adhesion: Loss of ROCK activity seems to lead to loss of tight junction integrity in endothelial cells. In epithelial cells inhibition of ROCK seems to decrease tight junction integrity. Active ROCK in these cells seems to stimulate the disruption of E-Cadherin-mediated cell-cell contacts by activating actomyosin contractility. 3. Other ROCK targets *NHE1 (a sodium hydrogen exchanger, involved in focal adhesions and actin organisation) *intermediate filament proteins: Vimentin, GFAP (glial fibrillaric acidic protein), NF-L (neurofilament L protein) *F-actin binding proteins: Adducin, EF-1&alpha (elongation factor, translation co-factor), MARCKS (myristylated alanine-rich C kinase substrate), Caponin (unknown function), and ERM (involved in linkage of the actin cytoskelton to the plasma membrane).

Homologues

The two mouse ROCK isoforms,

and ROCK2, have high homology. They have 65%

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...

sequences in common and 92% homology within their kinase domains. ROCKs are homologous to other metazoan kinases such as myotonic dystrophy kinase ( DMPK), DMPK-related cell division control protein 42 ( Cdc42)-binding kinases (MRCK) and citron kinase. All of these kinases are composed of a N-terminal kinase domain, a coiled-coil structure and other functional motifs at the C-terminus

Regulation

ROCK is a downstream effector molecule of the Rho GTPase Rho that increases ROCK kinase activity when bound to it. Autoinhibition ROCK activity is regulated by the disruption of an intramolecular autoinhibition. In general, the structure of ROCK proteins consists of an N-terminal kinase domain, a coiled-coiled region and a PH domain containing a cystein-rich domain (CRD) at the C-terminal. A Rho-binding domain (RBD) is located in close proximity just in front of the PH domain. The kinase activity is inhibited by the intramolecular binding between the C-terminal cluster of RBD domain and the

PH domain In chemistry, pH (), historically denoting "potential of hydrogen" (or "power of hydrogen"), is a scale used to specify the acidity or basicity of an aqueous solution. Acidic solutions (solutions with higher concentrations of ions) are ...

to the N-terminal kinase domain of ROCK. Thus, the kinase activity is off when ROCK is intramolecularly folded. The kinase activity is switched on when Rho-GTP binds to the Rho-binding domain of ROCK, disrupting the autoinhibitory interaction within ROCK, which liberates the kinase domain because ROCK is then no longer intramolecularly folded. Other regulators It has also been shown that Rho is not the only activator of ROCK. ROCK can also be regulated by lipids, in particular arachidonic acid, and protein

oligomerization In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relativ ...

, which induces N-terminal transphosphorylation.

Inhibitors

Disease

Research over the past two decades has shown that ROCK signaling plays an important role in many diseases including cardiovascular disease, neurodegenerative diseases such as Alzheimer's disease,

Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

, and amyotrophic lateral sclerosis, and

cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...

. For example, ROCK has been hypothesized to play an important role in the pleiotropic effects of statins. ROCK1/2 along with MRCKα/β kinases have been implicated in the plasticity of cancer cell migration, the phenomenon which bestows survival advantage to the cancer cells during drug treatments (

drug resistance Drug resistance is the reduction in effectiveness of a medication such as an antimicrobial or an antineoplastic in treating a disease or condition. The term is used in the context of resistance that pathogens or cancers have "acquired", that is ...

). Researchers are developing ROCK inhibitors such as RKI-1447 for treating various diseases including cancer. For example, such drugs have potential to prevent cancer from spreading by blocking cell migration, stopping cancer cells from spreading into neighboring tissue.

References