Narito Morii

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.

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.

Atrial natriuretic polypeptide: Topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry

The widespread distribution of neurons containing alpha-atrial natriuretic polypeptide-like immunoreactivity in the rat brain was demonstrated using radioimmunoassay and immunohistochemistry in conjunction with specific antisera. The highest concentrations of alpha-atrial natriuretic polypeptide-like immunoreactivity were in the hypothalamus and septum, with low but still appreciable concentrations in the mesencephalon, cerebral cortex, olfactory bulb and thalamus by radioimmunoassay. Immunohistochemical studies clearly showed that the perikarya of immunoreactive neurons are most prevalent in the ventral part of the lateral septal nucleus, periventricular preoptic nucleus, bed nucleus of the stria terminalis, periventricular and dorsal parts of the paraventricular hypothalamic nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, median mamillary nucleus, supramamillary nucleus, zona incerta, medial habenular nucleus and the periaqueductal grey matter. Scattered neurons were seen in the cingulate cortex, endopiriform nucleus, lateral hypothalamic area, and pretectal and dorsal thalamic areas. In addition to the areas mentioned above, high concentrations of immunoreactive varicose fibers were seen in the glomerular layer of the olfactory bulb, external layer of the median eminence, central to paramedian parts of the interpeduncular nucleus and the paraventricular hypothalamic nucleus. The globus pallidus, medial and central amygdaloid nuclei, dorsal raphe, dorsal parabrachial nucleus, locus coeruleus, vagal dorsal motor nucleus, solitary nucleus and some circumventricular organs, including the subfornical organ and organum vasculosum laminae terminalis, contained considerable numbers of immunoreactive varicose fibers. In dehydrated rats and homozygous Brattleboro rats, the pattern of alpha-atrial natriuretic polypeptide-immunoreactive neurons and varicose fibers was qualitatively similar to that seen in normal conditioned rats. This study gives an atlas of the distribution of the alpha-atrial natriuretic polypeptide-containing neuronal system in the rat brain and provides the groundwork for studying the influence of this new peptide on various brain functions.

α-Human atrial natriuretic polypeptide is released from the heart and circulates in the body

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.

Occurrence of atrial natriuretic polypeptide in brain

In order to determine whether or not atrial natriuretic polypeptides (ANPs) exist in the brain, we have studied extracts from the rat brain using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide. The presence and widespread distribution of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) have been demonstrated in the rat brain. The highest concentration of alpha-rANP-LI (20-22 ng/g) is in the hypothalamus and the septum. Moderate concentrations of alpha-rANP-LI (2-8 ng/g) are also found in the midbrain, cerebral cortex, olfactory bulb, thalamus, pons-medulla and hippocampus. High performance gel permeation chromatography coupled with the RIA revealed that alpha-rANP-LI found in the rat brain consists of three components eluting at the positions of gamma-rat ANP (gamma-rANP), beta-rat ANP (beta-rANP) and alpha-rANP, respectively. Among these a low molecular weight form of alpha-rANP-LI emerging at the elution position corresponding to alpha-rANP is predominant in the rat brain. This is in contrast to the finding that gamma-rANP, a high molecular weight form of 13k daltons is the dominant form of alpha-rANP-LI in the rat atrium. These results clearly show that there exists a widespread neural system containing ANPs in the brain.

A novel partner for the GTP-bound forms of rho and rac.

Using the yeast two hybrid system and overlay assays we identified a putative rho/rac effector, citron, which interacts with the GTP‐bound forms of rho and rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C6H2 zinc finger, a PH domain, and a long coiled‐coil forming region including 4 leucine zippers and the rho/rac binding site. We recently identified three others putative rho effectors characterized by a common rho binding motif. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners.

Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin

In this study we examined the role of rho p21 in neuropeptide‐stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin‐ and endothelin‐stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

Radioimmunoassay for α-human and rat atrial natriuretic polypeptide

Abstract Using a synthetic common carboxy-terminal fragment of α-human atrial natriuretic polypeptide (α-hANP) and α-rat atrial natriuretic polypeptide (α-rANP), we have produced an antiserum for α-ANP(17–28) and established a radioimmunoassay (RIA) for α-ANP that recognizes α-hANP and α-rANP equally. High performance gel permeation chromatography coupled with the RIA revealed that α-hANP-like immunoreactivity (α-hANP-LI) in the human atrium consists of three major components, γ-hANP, α-hANP and another. On the other hand, α-rANP-LI in the rat atrium comprised at least two components, 13K α-rANP-LI and 3K–5K α-rANP-LI, which were presumably γ-rANP and β-rANP, respectively. Thus, considerable amounts of γ-hANP and γ-rANP are present in human right auricles and rat atria, respectively. The RIA established in this study provides a useful tool to investigate the pathophysiological significance of α-ANP and related peptides in cardiovascular disorders and shows that γ-ANP is not only a precursor of α-ANP but acts as an important hormone.

ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells.

Botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) was purified to homogeneity and added to cultured rat pheochromocytoma PC-12 cells. Incubation with this exoenzyme caused inhibition of cell growth and induced neurites as well as acetylcholine esterase in these cells. These changes were dependent on the amount of the enzyme added to the culture, which correlated with the in situ ADP-ribosylation of the rho/rac proteins in the cells. Preincubation with a specific anti-C3 exoenzyme monoclonal antibody inhibited both the ADP-ribosyltransferase activity and the neurite-inducing activity of the enzyme preparation. These results suggest that C3 exoenzyme affected the cellular function of the rho/rac proteins by ADP-ribosylation to induce these changes in the cells.