Kazuko Fujisawa

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.

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.

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.

RHOTEKIN, A NEW PUTATIVE TARGET FOR RHO BEARING HOMOLOGY TO A SERINE/THREONINE KINASE, PKN, AND RHOPHILIN IN THE RHO-BINDING DOMAIN

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese “teki,” meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5′-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares ~30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

Different Regions of Rho Determine Rho-selective Binding of Different Classes of Rho Target Molecules

Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23–40 and 75–92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.

Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells

Lysophosphatidic acid (LPA) added to serum‐starved Swiss 3T3 cells induced, in a time‐ and concentration‐dependent manner, tyrosine phosphorylation of multiple proteins, including proteins of 43, 64, 88 kDa and a group of proteins between 110 and 130 kDa. Among them, two proteins, p43 and p120, were identified as mitogen‐activated protein kinase (MAP‐kinase) and focal adhesion kinase (FAK), respectively, by immunoprecipitation and immunoblot analysis. Tyrosine phosphorylation of p64 peaked at l min and declined rapidly, whereas that of MAP‐kinase and FAK peaked at 5 and 10 min after the addition of LPA, respectively. The activity of MAP‐kinase determined as phosphorylation of myelin basic protein increased transiently about 3‐fold at 5 min, and correlated with tyrosine phosphorylation. These results indicate that tyrosine phosphorylation of these proteins is a part of the signal transduction by LPA and may be involved in its mitogenic responses.

The Slit Receptor EVA-1 Coactivates a SAX-3/Robo–Mediated Guidance Signal in C. elegans

The SAX-3/roundabout (Robo) receptor has Shiga-like toxin 1 (SLT-1)/Slit–dependent and –independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit–dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.

Lysophosphatidic acid-induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts. Possible involvement in rho p21-mediated signalling.

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3Y1 fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64–65K (a doublet), and 60K (each named p145, p85, p64165 and p60, respectively) in addition to p43 mitogen activated protein (MAP)‐kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin‐insensitive rho p21‐dependent pathway and that the activation of MAP‐kinase was mediated by both the pertussis toxin‐sensitive rho p21‐independent and the pertussis toxin‐insensitive rho p21‐dependent pathways.