Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6.

Neurosphere cultures are widely used to propagate multipotent CNS precursors, but their differentiation into neurons or oligodendrocytes is rather poor. To elucidate fate determination in this system, we examined the expression and function of candidate transcription factors in neurospheres derived from different CNS regions during development and adulthood. We observed prominent down-regulation of most transcription factors present in telencephalic precursors upon growth factor exposure in neurosphere cultures while Olig1 and Olig2 expression was strongly up-regulated. Interference with Olig2 in neurospheres revealed its role in self-renewal during expansion and for the generation of neurons and oligodendrocytes during differentiation. We further show that neurogenesis becomes fully Pax6-dependent in the neurosphere culture system, independent of the region of origin, and that Pax6 overexpression is sufficient to direct almost all neurosphere-derived cells towards neurogenesis. Thus, a pathway combining transcription factors of dorsal and ventral regions is activated in the neurosphere culture model.

Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

Identification of sonic hedgehog-responsive genes using cDNA microarray.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

Proliferation of parenchymal neural progenitors in response to injury in the adult rat spinal cord.

It has long been believed that the fully developed mammalian central nervous system (CNS) lacks significant regenerative capacity. Recent advances have revealed, however, that many regions of the adult CNS contain neural progenitors that have the ability to generate new neurons and glia. Although the periventricular area has been identified as a rich source of these progenitors, their precise location in each region and details of their properties in vivo still remain poorly understood. Here we provide evidence that in the adult rat spinal cord, a significant number of neural progenitors are present, not only in the periventricular area, but also in other regions of the parenchyma. These progenitors could proliferate in vitro as neurosphere-like cell aggregates in the presence of growth factors and also gave rise to neurons and glia under appropriate conditions. We further demonstrate that these parenchymal neural progenitors were capable of proliferating in vivo in response to injury. Immunohistochemical studies suggested that proliferative progenitors emerged throughout the gray and white matter in the lesioned spinal cord. Consistently, an increased number of neurosphere-forming cells could be isolated from injured tissues, and they were able to differentiate into neurons in vitro. The widespread occurrence of neural progenitors in the parenchyma expands the possibility of repairing damaged tissue by activating the latent regenerative potential of the adult spinal cord.

Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons.

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA.

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U(n)AGU (n = 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3'-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3'-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis.

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-beta, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix-loop-helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.

Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1.

The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species [1]; defects in this pathway in humans can cause developmental disorders as well as neoplasia [2]. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood [3] [4]. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins [5] [6], including Costal2, Fused (Fu), and Suppressor of fused (Su(fu)), the last of which has been shown to interact directly with Ci [7]. We have cloned mouse Suppressor of fused (mSu(fu)) and detected its 4.5 kb transcript throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibited transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicated that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity were mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 was localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) resulted in a striking increase in cytoplasmic Gli1 immunostaining. Our findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors.

Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling.

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.

Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3.

Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to hedgehog protein (Hh) signaling. In the anterior region, in contrast, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products (GLIs) 1, 2, and 3) were identified, but their functional difference in Hh signal transduction is unknown. Here, we report distinct roles for GLI1 and GLI3 in Sonic hedgehog (Shh) signaling. GLI3 containing both repression and activation domains acts both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. The trans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to the Gli1 promoter and induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the activation of the target gene Gli1.

Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system.

Like other tissues and organs in vertebrates, multipotential stem cells serve as the origin of diverse cell types during genesis of the mammalian central nervous system (CNS). During early development, stem cells self-renew and increase their total cell numbers without overt differentiation. At later stages, the cells withdraw from this self-renewal mode, and are fated to differentiate into neurons and glia in a spatially and temporally regulated manner. However, the molecular mechanisms underlying this important step in cell differentiation remain poorly understood. In this study, we present evidence that the expression and function of the neural-specific transcription factors Mash-1 and Prox-1 are involved in this process. In vivo, Mash-1- and Prox-1-expressing cells were defined as a transient proliferating population that was molecularly distinct from self-renewing stem cells. By taking advantage of in vitro culture systems, we showed that induction of Mash-1 and Prox-1 coincided with an initial step of differentiation of stem cells. Furthermore, forced expression of Mash-1 led to the down-regulation of nestin, a marker for undifferentiated neuroepithelial cells, and up-regulation of Prox-1, suggesting that Mash-1 positively regulates cell differentiation. In support of these observations in vitro, we found specific defects in cellular differentiation and loss of expression of Prox-1 in the developing brain of Mash-1 mutant mice in vivo. Thus, we propose that induction of Mash-1 and Prox-1 is one of the critical molecular events that control early development of the CNS.

Pax-6 is involved in the specification of hindbrain motor neuron subtype.

Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2 exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos. By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.

A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro.

The floor plate plays important roles in ventral pattern formation and axonal guidance within the neural tube of vertebrate embryos. A critical event for floor plate development is the induction of a winged helix transcription factor, Hepatocyte Nuclear Factor-3beta (HNF-3beta). The enhancer for floor plate expression of HNF-3beta is located 3' of the transcription unit and consists of multiple elements. HNF-3beta induction depends on the notochord-derived signal, Sonic hedgehog (Shh). Genetic analysis in Drosophila has led to the identification of genes involved in the Hh signalling pathway, and cubitus interruptus (ci), encoding a protein with five zinc finger motifs, was placed downstream. In the present work, we test the involvement of Gli proteins, the mouse homologues of Ci, in activation of the floor plate enhancer of HNF-3beta. Transgenic analysis shows that a Gli-binding site is required for the activity of the minimal floor plate enhancer of HNF-3beta in vivo. Three Gli genes are differentially expressed in the developing neural tube. Gli expression is restricted to the ventral part, while Gli2 and Gli3 are expressed throughout the neural tube and dorsally, respectively. Strong Gli and Gli2, and weak Gli3 expressions transiently overlap with HNF-3beta at the time of its induction. Consistent with ventrally localized expression, Gli expression can be up-regulated by Shh in a cell line. Finally, the Gli-binding site acts as a Shh responsive element, and human GLI, but not GLI3, can activate this binding site in tissue culture. Taken together, these findings suggest that Gli, and probably also Gli2, are good candidates for transcriptional activators of the HNF-3beta floor plate enhancer, and the binding site for Gli proteins is a key element for response to Shh signalling. These results also support the idea that Gli/Ci are evolutionary conserved transcription factors in the Hedgehog signalling pathway.

Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1.

Cdc42 and Rac1 have been implicated in the regulation of various cell functions such as cell morphology, polarity, and cell proliferation. We have partially purified a Cdc42- and Rac1-associated protein with molecular mass of about 170 kDa (p170) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS++.GST-Rac1 but not with the GDP.GST-Cdc42, GDP.GST-Rac1, or GTPgammaS.GST-RhoA). We identified p170 as an IQGAP, which is originally identified as a putative Ras GTPase-activating protein. Recombinant IQGAP specifically interacted with GTPgammaS.Cdc42 and GTPgammaS.Rac1. The C-terminal fragment of IQGAP was responsible for their interactions. IQGAP was specifically immunoprecipitated with dominant-active Cdc42(Val12) or Rac1(Val12) from the COS7 cells expressing Cdc42(Val12) or Rac1(Val12), respectively. Immunofluorescence analysis revealed that IQGAP was accumulated at insulin- or Rac1-induced membrane ruffling areas. This accumulation of IQGAP was blocked by the microinjection of the dominant-negative Rac1(Asn17) or Cdc42(Asn17). Moreover, IQGAP was accumulated at the cell-cell junction in MDCK cells, where alpha-catenin and ZO-1 were localized. These results suggest that IQGAP is a novel target molecule for Cdc42 and Rac1.

Biosynthesis and expression of polysialic acid on the neural cell adhesion molecule is predominantly directed by ST8Sia II/STX during in vitro neuronal differentiation.

We have reported recently that ST8Sia II/STX as well as ST8Sia IV/PST-1 is a neural cell adhesion molecule (NCAM)-specific polysialic acid (PSA) synthase (Kojima, N., Tachida, Y., Yoshida, Y., and Tsuji, S. (1996) J. Biol. Chem. 271, 19457-19463). To investigate which of two PSA synthase (ST8Sia II and IV) are involved in the biosynthesis of PSA associated with NCAM, the expressions of PSA, PSA synthase activity, and the genes of two PSA synthases during in vitro neuronal differentiation of mouse embryonal carcinoma P19 cells were determined. PSA was not expressed on undifferentiated cells (day 0) or cell aggregates (days 1-3) induced with retinoic acid. Expression of PSA began after cell aggregates had been dissociated and re-plated on a dish (day 4) and increased up to day 7. The expression of the mouse ST8Sia II gene was negligible in both undifferentiated and aggregated cells, it beginning at day 4, then dramatically increasing, and reaching the maximum level at days 6-7. On the other hand, transcription of the ST8Sia IV gene remained at a very low level throughout the entire period, a significant increase in its expression during differentiation not being observed. PSA synthase activity was not detected in undifferentiated or aggregated P19 cells, it increasing in parallel with ST8Sia II gene expression during differentiation. In addition, the cells at day 7 were stained with an anti-mouse ST8Sia II antiserum. Similar up-regulation of the ST8Sia II gene were observed during the differentiation of rat MNS-8 cells, which were derived from E-12 rat neuroepithelium of the neural tube and shown to differentiate into neurons. These results indicate that ST8Sia II predominantly directs PSA expression during neuronal differentiation rather than ST8Sia IV.

Roles of cell-autonomous mechanisms for differential expression of region-specific transcription factors in neuroepithelial cells.

Although a number of genes have been found to have restricted expression domains in the embryonic forebrain and midbrain, it remains largely unknown how the expression of these genes is regulated at the cellular level. In this study, we explored the mechanisms for the differential expression of region-specific transcription factors in neuroepithelial cells by using both primary and immortalized neuroepithelial cells from the rat brain at embryonic day 11.5. We found that differential expression patterns of Pax-3, Pax-5, Pax-6, Dlx-1, Dlx-2, Emx2, Otx1 and Dbx observed in vivo were maintained even when the cells were isolated and cultured in vitro, free from environmental influences. Furthermore, in response to Sonic hedgehog, which is a major inductive signal from the environment for regional specification, neuroepithelial cells that maintain distinct regional identities expressed different sets of ventral-specific genes including Islet-1, Nkx-2.1 and Nkx-2.2. These results suggest that certain cell-autonomous mechanisms play important roles in regulating both environmental signal-dependent and -independent expression of region-specific genes. Thus, we propose that use of the in vitro culture systems we describe in this study facilitates the understanding of regulatory mechanisms of region-specific genes in neuroepithelial cells.

Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase)

The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.