Toshimasa Ishizaki

Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension.

Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP‐binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP‐bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP‐bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N‐terminus, followed by a coiled‐coil structure approximately 600 amino acids long, and a cysteine‐rich zinc finger‐like motif and a pleckstrin homology region in the C‐terminus. The N‐terminus region including a kinase domain and a part of coiled‐coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co‐expressed with RhoA in COS cells, p160 was co‐precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Cooperation between mDia1 and ROCK in Rho-induced actin reorganization

The small GTPase Rho induces the formation of actin stress fibres and mediates the formation of diverse actin structures. However, it remains unclear how Rho regulates its effectors to elicit such functions. Here we show that GTP-bound Rho activates its effector mDia1 by disrupting mDia1’s intramolecular interactions. Active mDia1 induces the formation of thin actin stress fibres, which are disorganized in the absence of activity of the Rho-associated kinase ROCK. Moreover, active mDia1 transforms ROCK-induced condensed actin fibres into structures reminiscent of Rho-induced stress fibres. Thus mDia1 and ROCK work concurrently during Rho-induced stress-fibre formation. Intriguingly, mDia1 and ROCK, depending on the balance of the two activities, induce actin fibres of various thicknesses and densities. Thus Rho may induce the formation of different actin structures affected by the balance between mDia1 and ROCK signalling.

p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin‐related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP‐bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co‐localized in the spreading lamellae of cultured fibroblasts. They are also co‐localized in membrane ruffles of phorbol ester‐stimulated sMDCK2 cells, which extend these structures in a Rho‐dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin‐coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS‐7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.

ROCK‐I and ROCK‐II, two isoforms of Rho‐associated coiled‐coil forming protein serine/threonine kinase in mice

We recently identified a novel human protein kinase, p160 ROCK, as a putative downstream target of the small GTPase Rho. Using the human ROCK cDNA as a probe, we isolated cDNA of two distinct, highly related sequences from mouse libraries. One encoded a mouse counterpart of human ROCK (ROCK‐I), and the other encoded a novel ROCK‐related kinase (ROCK‐II). Like ROCK/ROCK‐I, ROCK‐II also bound to GTP‐Rho selectively. ROCK‐I mRNA was ubiquitously expressed except in the brain and muscle, whereas ROCK‐II mRNA was expressed abundantly in the brain, muscle, heart, lung and placenta. These results suggest that at least two ROCK isoforms are present in a single species and play distinct roles in Rho‐mediated signalling pathways.

p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions

© 1997 Federation of European Biochemical Societies.

Rho effectors and reorganization of actin cytoskeleton

The small GTPase Rho regulates several actomyosin‐based cellular processes such as cell adhesion, cytokinesis and contraction. The biochemical mechanisms of these actions remain unknown. Recently, several GTP‐Rho binding proteins were isolated. Among them, p140mDia and p160ROCK appear to work as Rho effectors mediating its action on the cytoskeleton. p140mDia induces actin polymerization by recruiting an actin binding protein, profilin, to the site of Rho action. p160ROCK induces focal adhesions and stress fibers by activating integrin and clustering them by the use of myosin‐based contractility.

Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion

The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.

Role of citron kinase as a target of the small GTPase Rho in cytokinesis

During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody,. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures,, is known to be essential. Protein kinases are also thought to participate in cytokinesis,,. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho

The Rho guanosine 5′-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.