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ROCK Signaling Pathway

Figure 1. ROCK signaling pathway.

ROCK overview

The Rho subfamily belongs to a member of the small molecule G protein in the Ras superfamily and has GTPase activity. It converts between an activated state (bound to GTP) and an inactive state (bound to GDP), acting as a molecular switch, and is a Rho protein that exerts various biological effects by binding to its downstream target effector molecule. Among them, Rho kinase (ROCK) is one of the most important effector molecules downstream of Rho. ROCK is a serine/threonine protein Kinase with a relative molecular mass of approximately 160000. There are two homologous isomers in the cell: ROCK1 (ROCK-I, ROKβ or p160 ROCK) and ROCK2 (ROCK-II, ROKα or Rho kinase). Rho/ROCK signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling by regulating actin and cell migration. Studies on the function of ROCK proteins have focused on the cardiovascular system and the central nervous system. Studies have found that abnormal expression of ROCK protein plays an important role in the development of tumors.

ROCK family

The ROCK1 and ROCK2 genes contain 33 exons, each located in the 18q11 and 2p24 chromosome regions. In addition to the full-length transcript, ROCK2 also has a splicing mutant (mROCK2). mROCK2 has an additional exon 27, and the 5th exon deleted ROCK1 pseudogene, called the small ROCK, appears to be a product of partial gene duplication and is expressed only in chromosome 18p11 of vascular smooth. The ROCK molecular structure consists of three parts, and the first part is the N-terminal kinase region, consisting of approximately 300 amino acid residues. The composition consists of a serine/threonine protein kinase-associated conserved sequence that catalyzes the phosphorylation/dephosphorylation of a series of downstream substrates; the second is a coiled-coil region located in the middle that interacts with other α-helix proteins. The Rho-binding domain (RBD) consisting of approximately 80 amino acid residues is included, which can accept the activation signal of Rho transduction, such an activating the phosphotransferase activity of ROCK; The third part is located at C-terminal. The pleckstrin homology domain (PHD) may be involved in protein localization, including the cysteine-rich domain (CRD), and the C-terminus contains a self-inhibiting region, and interacts with the kinase domain to inhibit ROCK activity. The full-length amino acid sequence homology of the two subtypes of ROCK1 and ROCK2 is 65%, and the homology of the kinase region is as high as 92%. The expression of ROCK protein is expressed from embryonic development to adult mouse tissues. ROCK1 mRNA and protein are highly expressed in lung, liver, spleen, kidney and testis, and ROCK2 is highly expressed in brain and heart. ROCK2 is mainly concentrated in the cytoplasm and is involved in vimentin and actin tensile fibers. It is also localized to the plasma membrane through the C-terminal region. The mROCK2 protein has a similar cellular localization to ROCK2, the only difference being that the mROCK2 protein is not associated with the plasma membrane. In contrast, the cell distribution of ROCK1 is not well understood. Numerous studies have shown that ROCK1 is primarily localized on the plasma membrane of endothelium. ROCK1 acts indirectly on the epithelial-cadherin complex by binding to the E-cadherin scaffold protein P120-catenin. In addition, ROCK1 can be concentrated in the center of the microtubule tissue and the pseudopod edge of the moving cells, indicating that ROCK1 is involved in cell migration.

Rock signaling pathway

  1. ROCK signaling pathway cascade
  2. The ROCK protein phosphorylates many downstream targets, such as changes in the structural composition of F-actin (filament actin), thereby affecting cell contractility, motility and morphology. ROCK1 and ROCK2 may have different upstream signals, but their substrates include myosin light chain (MLC) LIM (Lin-11 Isl-1 Mec-3) Kinase (LIMK), Ezrin/Radixin/Moesin (ERM) and intermediate filament proteins. In most cases, substrates are phosphorylated by their respective ROCK proteins, but homologous family members, especially homologous to the kinase region, can be phosphorylated by the ROCK protein. Most of the substrates are phosphorylated on amino acid-specific sequences, like K/RXS/T or K/R-XX-S/T (X is any amino acid), therefore, ROCK substrates are divided into three groups: (1) actin-related; (2) intermediate filament proteins; (3) others. Myosin Light Chain: Myosin II is a multi-subunit unit protein complex with catalytic activity that regulates myogenesis and myosin-mediated cell contraction. Both MLC1 and 2 are core proteins of the myosin II catalytic domain, which regulate myosin ATPase activity, thereby interfering with filamentous actin and regulating cell contraction. Phosphorylation of MLC2 enhances ATPase activity of myosin II and enhances cell shrinkage, whereas phosphorylation is primarily dependent on Ca2+, a balance between MLC kinase (MLCK) and MLC phosphatase (MYPT). Recently, there is increasing evidence that phosphorylation of MLC can be phosphorylated by ROCK2 independent of Ca2+ concentration, but MLCK is still dominant in the absence of Ca2+, and activation of Rho-ROCK will only result in a small amount of MLC. Myosin-binding subunit: MYPT is a heterotrimeric complex that regulates MYPT targeting to the myosin binding subunit (MBS), a 1c subunit with phosphatase catalytic activity, and an unknown function of the M20 subunit. In early in vitro studies, MBS was considered a substrate for ROCK2. The phosphorylation of MBS inhibits the catalytic activity of MYPT, thereby inducing phosphorylation of MLC and myosin ATP enzyme activity, therefore, the ROCK signaling pathway doubles phosphorylation of MLC by direct phosphorylation and inhibition of its dephosphorylation. LIMK family members include LIMK1 and LIMK2, both of which are serine/threonine kinases. Phosphorylation and inhibition of actin depolymerization factor cofilin activity regulates actin movement. ROCK1 and ROCK2 phosphorylate LIMK1 and LIMK2 at Thr 508 and Thr 505, respectively, to facilitate their activation. Upon their activation, LIMKs phosphorylate cofilin at a highly conserved serine 3 site, preventing its binding to F-actin, inhibiting actin depolymerization and actin filament breaks, and promoting intracellular enhancement of F-actin network structure. LIMKs can also be activated by the p-21-activated kinases PAK1 and PKA4, which are downstream target proteins of the Rho/GTPases Rac and Cdc42. The ERM complex is an anchoring protein linked to F-actin and the plasma membrane, which plays an important role in the rapid movement of actin and cytoskeletal rearrangement. In vitro cell research, radixin, moesin, and ezrin (Ezrin) are substrates of ROCK2, suggesting that ROCK2 phosphorylates ERM complexes and enhances their interaction with intracellular molecules and redisperses them on the plasma membrane to enhance ERM activity. Of course, ERM can also be phosphorylated by other kinases, including protein kinase C-theta, DMPK-associated MRCK, and lymphocyte localization kinase. ERM phosphorylation leads to an increase in the affinity of the trimer, resulting in reduced cell migration. MYPT, one of the ROCK substrates, also exhibits phosphatase activity against the membrane protein, so it is speculated that ROCK can also phosphorylate ERM, thereby causing an increase in ERM activity and playing an important role in regulating the cytoskeleton.

  3. Pathway regulation
  4. The C-terminus of the ROCK protein is a self-inhibiting region of kinase activity, including the RBD region and the PH region. Both regions independently bind to the N-terminal kinase region, thereby inhibiting kinase activity. In the inactive state, the RBD and PH regions of the C-terminus of the ROCK protein interact with the kinase domain to form a self-inhibiting loop, truncating its C-terminus (RBD and PH regions) and allowing ROCK to be continuously activated. Consistent with this, the expression of a different C-terminal structure or a deletion region of the kinase region on the full-length ROCK molecule indicates a dominant inactivation of the ROCK protein function. The above evidence indicates that the C-terminus of the ROCK protein mainly plays an inhibitory role in the cell. The positive regulation of ROCK protein activity is mainly in the following three ways: (1) The activated state of RhoA (coupled with GTP) binds to the RBD region of ROCK, which changes the configuration of ROCK and interferes with the C-terminal inhibition domain of ROCK protein. The kinase domain interacts to abolish the inhibition of ROCK at the C-terminus and activates ROCK. Some lipids interact with the PH region of the C-terminal inhibitory region of ROCK protein, interferes with the C-terminal inhibitory activity, and thus activates ROCK without relying on RhoA activity. For example, sphingosylphosphorylcholine (SPC) interactions in the PH region result in a significant increase in ROCK activity. The cleavage of the inhibitory C-terminus from ROCK also leads to constitutive activation of the ROCK protein. In the process of apoptosis, capsase-3 cleaves ROCK1 at DETD1113, resulting in activation of ROCK1. This capsase-3 cleavage site is not present on ROCK2; apoptotic protease granzyme B cleaves ROCK2 at the IGLD1131 site, removing its inhibitory region to activate ROCK2, and this granzyme B cleavage site is not present on ROCK1. There are also three main ways of negative regulation of ROCK activity. Some small molecule G proteins have a negative regulation effect on ROCK activity.

  5. Relationship with disease
  6. Tumor

    As one of the most important downstream target molecules of the Rho family, ROCK protein activates myosin, cross-links it, and induces actomy and contraction of actomyosin, thereby participating in the regulation of cell morphology, polarity, cytoskeletal remodeling and cell migration. Many studies have shown that ROCK protein plays an important role in tumorigenesis and development, and ROCK expression is increased in osteosarcoma, hepatocellular carcinoma and skin cancer tissues. Moreover, many substrates of ROCK protein play a significant role in tumor development and related phenotypes, while ROCK inhibitors can inhibit the migration of a variety of tumor cell lines. ROCK protein is involved in the regulation of tumor cell survival and apoptosis, and malignant tumor cell invasion and metastasis.

    Diabetic nephropathy

    High glucose activates the Rho/ROCK signaling pathway in mesangial cells, activates the transcription factor AP-1, up-regulates fibronectin, and leads to accumulation of glomerular matrix proteins. Activation of the Rho/ROCK signaling pathway regulates the NF-κB signaling pathway, upregulates inflammatory genes and induces the development of diabetic nephropathy. ROCK inhibitors can reduce the sclerosing cytokines and extracellular matrix, resist oxidation and protect mitochondria, thereby reducing the rate of progression of sclerosis and fibrosis in diabetic nephropathy, inhibiting glomerular permeability and protecting the kidneys.

References

  1. Yamashita F, Fukuyama E, Mizoguchi K, et al. Scale dependence of rock friction at high work rate. Nature. 2015, 528(7581):254-257.
  2. Shimizu Y, Dobashi K, Sano T, et al. ROCK activation in lung of idiopathic pulmonary fibrosis with oxidative stress. International Journal of Immunopathology & Pharmacology. 2014, 27(1):37.
  3. Zhang C, Zhang S, Zhang Z, et al. ROCK has a crucial role in regulating prostate tumor growth through interaction with c-Myc. Oncogene. 2014, 33(49):5582-5591.
  4. Alleaumebutaux A, Nicot S, Pietri M, et al. Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation. Plos Pathogens. 2015, 11(8):1005073.

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