Rho-associated protein kinase
Encyclopedia
Rho-associated protein kinase (ROCK) is a kinase
belonging to the AGC (PKA/ PKG/PKC) family of serine-threonine kinases
. It is mainly involved in regulating the shape and movement of cells by acting on the cytoskeleton
.
ROCKs (ROCK1 and ROCK2) occur in mammals (human, rat, mouse, cow), zebrafish, Xenopus, invertebrates (C. elegans, Mosquito, Drosophila) and chicken. Human ROCK1 has a molecular mass
of 158 kDa
and is a major downstream effector of the small GTPase
RhoA
. Mammalian ROCK consists of a kinase domain, a coiled-coil region and a Pleckstrin homology (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
s and focal adhesions by phosphorylating MLC
(myosin light chain)
.
Due to this phosphorylation
, the actin
binding of myosin II and, thus, the contractility
increases. Two mouse ROCK isoforms ROCK1
and ROCK2 have been identified. ROCK1 is mainly expressed in the lung
, liver
, spleen
, kidney
and testis. However, ROCK2 is distributed mostly in the brain
and heart
.
Rho
, which is one of the major regulators of the cytoskeleton
.
1. ROCK is a key regulator of actin organization and thus a regulator of cell migration
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 organisation and contractility as follows:.
ROCK inhibits the depolymerisation of actin filaments indirectly: ROCK phosphorylates and activates LIM kinase, 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 .
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:
Thus in both cases, ROCK activation by Rho induces the formation of actin stress fibres, actin filament bundles of opposing polarity, containing myosin II, tropomyosin, caldesmon and MLC-Kinase, and consequently of focal contacts, which are immature integrin-based adhesion points with the extracellular substrate .
2. Other functions and targets
3. Other ROCK targets
and ROCK2 have high homology
. They have 65% amino acid
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 theses kinases are composed of a N-terminal kinase domain, a coiled-coil structure and other functional motifs
at the C-terminus
Rho which increases ROCK kinase activity when bound to it.
Autoinhibition
ROCK activity is regulated by the disruption of an intramolecular autoinhibition. The structure of ROCK proteins generally 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 to the N-terminal kinase domain of ROCK. Thus the kinase activity is off when ROCK is intramoleculary 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 which induces N-terminal transphosphorylation.
and cancer
. It has been shown to be involved in causing tissue thickening and stiffening around tumours in a mouse model of skin cancer, principally by increasing the amount of collagen
in the tissue around the tumour.
Researchers are developing ROCK inhibitors
for treating disease. For example, such drugs could potentially prevent cancer from spreading by blocking cell migration, stopping cancer cells from spreading into neighbouring tissue.
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
belonging to the AGC (PKA/ PKG/PKC) family of serine-threonine kinases
Serine/threonine-specific protein kinase
Serine/threonine protein kinases phosphorylate the OH group of serine or threonine .At least 125 of the 500+ human protein kinases are serine/threonine kinases .-Regulation:...
. It is mainly involved in regulating the shape and movement of cells by acting on the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
.
ROCKs (ROCK1 and ROCK2) occur in mammals (human, rat, mouse, cow), zebrafish, Xenopus, invertebrates (C. elegans, Mosquito, Drosophila) and chicken. Human ROCK1 has a molecular mass
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
of 158 kDa
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
and is a major downstream effector of the small GTPase
GTPase
GTPases are a large family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate . The GTP binding and hydrolysis takes place in the highly conserved G domain common to all GTPases.-Functions:...
RhoA
RHOA
Ras homolog gene family, member A is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. In humans, it is encoded by the gene RHOA....
. Mammalian ROCK consists of a kinase domain, a coiled-coil region and a Pleckstrin homology (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 fiber
Stress fibers are high order structures in cells consisting of actin filaments , crosslinking proteins , and myosin II motors.Actin is a globular protein...
s and focal adhesions by phosphorylating MLC
Myosin
Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
(myosin light chain)
.
Due to this phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
, the actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
binding of myosin II and, thus, the contractility
Contractility
Myocardial contractility is the intrinsic ability of the heart to contract independent of preload and afterload. Changes in the ability to produce force during contraction result from different degrees of binding between myosin and actin filaments...
increases. Two mouse ROCK isoforms ROCK1
ROCK1
Rho-associated, coiled-coil containing protein kinase 1, also known as ROCK1, is a protein serine/threonine kinase, but also denotes the human gene encoding it....
and ROCK2 have been identified. ROCK1 is mainly expressed in the lung
Lung
The lung is the essential respiration organ in many air-breathing animals, including most tetrapods, a few fish and a few snails. In mammals and the more complex life forms, the two lungs are located near the backbone on either side of the heart...
, liver
Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion...
, spleen
Spleen
The spleen is an organ found in virtually all vertebrate animals with important roles in regard to red blood cells and the immune system. In humans, it is located in the left upper quadrant of the abdomen. It removes old red blood cells and holds a reserve of blood in case of hemorrhagic shock...
, kidney
Kidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...
and testis. However, ROCK2 is distributed mostly in the brain
Brain
The brain is the center of the nervous system in all vertebrate and most invertebrate animals—only a few primitive invertebrates such as sponges, jellyfish, sea squirts and starfishes do not have one. It is located in the head, usually close to primary sensory apparatus such as vision, hearing,...
and heart
Heart
The heart is a myogenic muscular organ found in all animals with a circulatory system , that is responsible for pumping blood throughout the blood vessels by repeated, rhythmic contractions...
.
Function
ROCK plays a role in a wide range of different cellular phenomena, as ROCK is a downstream effector protein of the small GTPaseGTPase
GTPases are a large family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate . The GTP binding and hydrolysis takes place in the highly conserved G domain common to all GTPases.-Functions:...
Rho
Rho
Rho is the 17th letter of the Greek alphabet. In the system of Greek numerals, it has a value of 100. It is derived from Semitic resh "head"...
, which is one of the major regulators of the cytoskeleton
Cytoskeleton
The cytoskeleton is a cellular "scaffolding" or "skeleton" contained within a cell's cytoplasm and is made out of protein. The cytoskeleton is present in all cells; it was once thought to be unique to eukaryotes, but recent research has identified the prokaryotic cytoskeleton...
.
1. ROCK is a key regulator of actin organization and thus a regulator of cell migration
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 directions to specific locations...
as follows:
Different substrates can be phosphorylated by ROCKs, including LIM kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
, myosin
Myosin
Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
light chain (MLC) and MLC phosphatase
Phosphatase
A phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group . This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their...
. These substrates, once phosphorylated, regulate actin filament organisation and contractility as follows:.
- Amount of actin filaments
ROCK inhibits the depolymerisation of actin filaments indirectly: ROCK phosphorylates and activates LIM kinase, which in turn phosphorylates ADF/cofilin
Cofilin
ADF/cofilin is a family of actin-binding proteins which disassembles actin filaments.Three highly conserved and highly identical genes belonging to this family have been described in human and mice:...
, 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
Contraction
Contraction may refer to:In physiology:* Muscle contraction, one that occurs when a muscle fiber lengthens or shortens** Uterine contraction, contraction of the uterus, such as during childbirth* Contraction, a stage in wound healing...
and thus cell-substratum contacts. ROCK increases the activity of the motor protein myosin
Myosin
Myosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
II by two different mechanisms:
- Firstly, phosphorylation of the myosin light chain (MLCMyosinMyosins comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes. They are responsible for actin-based motility. The term was originally used to describe a group of similar...
) increases the myosin II ATPaseATPaseATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
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 phosphatasePhosphataseA phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group . This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their...
, leading to increased levels of phosphorylated MLC.
Thus in both cases, ROCK activation by Rho induces the formation of actin stress fibres, actin filament bundles of opposing polarity, containing myosin II, tropomyosin, caldesmon and MLC-Kinase, and consequently of focal contacts, which are immature integrin-based adhesion points with the extracellular substrate .
2. Other functions and targets
- RhoA-GTP stimulates the phospholipid phosphatase activity of PTENPTEN (gene)Phosphatase and tensin homolog is a protein that, in humans, is encoded by the PTEN gene. Mutations of this gene are a step in the development of many cancers....
(phosphatasePhosphataseA phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group . This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their...
and tensin homologue), a human tumorTumorA tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
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 G1, and is proposed to be required for contraction of the cleavage furrow, which is necessary for the completion of cytokinesisCytokinesisCytokinesis is the process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells. It usually initiates during the late stages of mitosis, and sometimes meiosis, splitting a binucleate cell in two, to ensure that chromosome number is maintained from one generation...
.
- ROCKs also seem to antagonize the insulinInsulinInsulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
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 apoptosisApoptosisApoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. 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 regulates also MLC phosphorylation and actomyosin contractility which regulate membrane blebbing. - ROCKs contribute to neuriteNeuriteA neurite 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 culture, because it can be difficult to tell axons from dendrites before...
retraction by inducing growth coneGrowth coneA growth cone is a dynamic, actin-supported extension of a developing axon seeking its synaptic target. Their existence was originally proposed by Spanish histologist Santiago Ramón y Cajal based upon stationary images he observed under the microscope...
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. However 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 ROCK1ROCK1
Rho-associated, coiled-coil containing protein kinase 1, also known as ROCK1, is a protein serine/threonine kinase, but also denotes the human gene encoding it....
and ROCK2 have high homology
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
. They have 65% amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
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
CDC42
Cell division control protein 42 homolog also known as CDC42 is a protein involved in regulation of the cell cycle. In humans, CDC42 is encoded by the CDC42 gene.- Function :...
)-binding kinases (MRCK) and citron kinase. All of theses kinases are composed of a N-terminal kinase domain, a coiled-coil structure and other functional motifs
Sequence motif
In genetics, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance...
at the C-terminus
Regulation
ROCK is a downstream effector molecule of the Rho GTPaseRho family of GTPases
The Rho family of GTPases is a family of small signaling G protein , 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 organisms including yeasts and some plants...
Rho which increases ROCK kinase activity when bound to it.
Autoinhibition
ROCK activity is regulated by the disruption of an intramolecular autoinhibition. The structure of ROCK proteins generally 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
Intramolecular
Intramolecular in chemistry describes a process or characteristic limited within the structure of a single molecule, a property or phenomenon limited to the extent of a single molecule.- Examples :...
binding between the C-terminal cluster of RBD domain and the PH domain to the N-terminal kinase domain of ROCK. Thus the kinase activity is off when ROCK is intramoleculary 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
Activator (genetics)
An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of transcription. The activator may increase transcription by virtue of a connected domain which assists in the formation of the RNA polymerase holoenzyme, or may operate through a coactivator. A...
of ROCK. ROCK can also be regulated by lipids, in particular arachidonic acid
Arachidonic acid
Arachidonic acid is a polyunsaturated omega-6 fatty acid 20:4.It is the counterpart to the saturated arachidic acid found in peanut oil, Arachidonic acid (AA, sometimes ARA) is a polyunsaturated omega-6 fatty acid 20:4(ω-6).It is the counterpart to the saturated arachidic acid found in peanut oil,...
, and protein oligomerization which induces N-terminal transphosphorylation.
Disease
Recent research has shown that ROCK signaling plays an important role in many diseases including diabetes, neurodegenerative diseases, pulmonary hypertensionPulmonary hypertension
In medicine, pulmonary hypertension is an increase in blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillaries, together known as the lung vasculature, leading to shortness of breath, dizziness, fainting, and other symptoms, all of which are exacerbated by exertion...
and cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
. It has been shown to be involved in causing tissue thickening and stiffening around tumours in a mouse model of skin cancer, principally by increasing the amount of collagen
Collagen
Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content...
in the tissue around the tumour.
Researchers are developing ROCK inhibitors
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
for treating disease. For example, such drugs could potentially prevent cancer from spreading by blocking cell migration, stopping cancer cells from spreading into neighbouring tissue.