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.
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
GTPaseRhoA. 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.[1][2]
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.[5] 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
GTPaseRho, 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 organization and contractility as follows:[2]
Amount of actin filaments
ROCK inhibits the depolymerization 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.[2][6]
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
stress fibers, 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][7]
2. Other functions and targets
RhoA-GTP stimulates the phospholipid phosphatase activity of
PTEN (
phosphatase and tensin homologue), a human
tumor suppressor protein. This stimulation seems to depend on ROCK.[8][9] 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
cytokinesis.[2][10][11][12][13][14]
ROCKs also seem to antagonize the
insulin signaling pathway, resulting in a reduction of cell size and influence cell fate.[2]
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.[2]
ROCKs contribute to
neurite retraction by inducing
growth cone 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.[2]
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.[2]
3. Other ROCK targets
NHE1 (a sodium hydrogen exchanger, involved in focal adhesions and actin organisation)
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
Rho-associated, coiled-coil-containing protein kinase 1
The two mouse ROCK isoforms,
ROCK1 and ROCK2, have high
homology. They have 65%
amino acid sequences in common and 92% homology within their kinase domains.
[1][4]
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 [2]
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 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.[2]
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.[2]
Researchers are developing
ROCK inhibitors such as
RKI-1447 for treating various diseases including cancer.[20][21] For example, such drugs have potential to prevent cancer from spreading by blocking cell migration, stopping cancer cells from spreading into neighboring tissue.[1]
^Anjum I (June 2018). "Calcium sensitization mechanisms in detrusor smooth muscles". Journal of Basic and Clinical Physiology and Pharmacology. 29 (3): 227–235.
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^Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S (August 1999). "Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase". Science. 285 (5429): 895–8.
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10.1126/science.285.5429.895.
PMID10436159.
^Li Z, Dong X, Dong X, Wang Z, Liu W, Deng N, Ding Y, Tang L, Hla T, Zeng R, Li L, Wu D (April 2005). "Regulation of PTEN by Rho small GTPases". Nature Cell Biology. 7 (4): 399–404.
doi:
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^Feng Y, LoGrasso PV, Defert O, Li R (March 2016). "Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential". Journal of Medicinal Chemistry. 59 (6): 2269–2300.
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