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.

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.

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.

Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase.

Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis.

Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow.

The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.

Distribution of Rho-kinase in the bovine brain.

Rho-associated kinase (Rho-kinase) is a serine/threonine protein kinase downstream of the small GTPase Rho, which participates in signaling pathways of many cellular functions. Although Rho-kinase is implicated in the regulation of the morphology of neuronal cells, the distribution of Rho-kinase in the brain has not been elucidated yet. In this study, we investigated the distribution of Rho-kinase using three antibodies recognizing the different epitopes of Rho-kinase. Rho-kinase was abundantly expressed in the gray matter in comparison with the white matter. Strong immunoreactivity was observed in the pyramidal neurons of the cerebral cortex and hippocampus and in the Purkinje cells of the cerebellum. These results indicate that Rho-kinase is abundantly distributed in neurons and might play an important role in remodeling of neurites.

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Phosphorylation of vimentin by Rho-associated kinase at a unique amino-terminal site that is specifically phosphorylated during cytokinesis.

We found that vimentin, the most widely expressed intermediate filament protein, served as an excellent substrate for Rho-associated kinase (Rho-kinase) and that vimentin phosphorylated by Rho-kinase lost its ability to form filaments in vitro. Two amino-terminal sites on vimentin, Ser38 and Ser71, were identified as the major phosphorylation sites for Rho-kinase, and Ser71 was the most favored and unique phosphorylation site for Rho-kinase in vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, we prepared an antibody GK71 that specifically recognizes the phosphorylation of vimentin-Ser71. Ectopic expression of constitutively active Rho-kinase in COS-7 cells induced phosphorylation of vimentin at Ser71, followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser71 occurred only at the cleavage furrow in late mitotic cells but not in interphase or early mitotic cells. This cleavage furrow-specific phosphorylation of vimentin-Ser71 was observed in the various types of cells we examined. All these accumulating observations increase the possibility that Rho-kinase may have a definite role in governing regulatory processes in assembly-disassembly and turnover of vimentin filaments at the cleavage furrow during cytokinesis.

Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN.

PKN is a serine/threonine protein kinase with a catalytic domain homologous to the protein kinase C family and unique N-terminal leucine zipper-like sequences. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that the regulatory domain of PKN interacted with vimentin. We then examined whether PKN would phosphorylate vimentin in vitro. Vimentin proved to be an excellent substrate for PKN, and the phosphorylation of vimentin by PKN occurred in the head domain with the result of a nearly complete inhibition of its filament formation in vitro. Similar results were also obtained with another type III intermediate filament protein, glial fibrillary acidic protein (GFAP). These results raise the possibility that PKN may regulate filament structures of vimentin and GFAP by domain-specific phosphorylation.

Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase.

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.

Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries.

MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.

Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes.

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.

Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest.

The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.

Characterization of recombinant Xenopus MAP kinase kinases mutated at potential phosphorylation sites.

Xenopus mitogen-activated protein kinase kinase (MAPKK) previously inactivated with protein phosphatase 2A can be reactivated by serine phosphorylation catalyzed by a partially purified MAPKK kinase (MAPKK-K), and is phosphorylated by MAPK on a threonine residue. The sequence analysis of a threonine-phosphorylated tryptic peptide of Xenopus MAPKK from mature oocytes suggested that Thr388 is phosphorylated in vivo. A mutant MAPKK that has Thr388 changed to Ala (T388A-MAPKK) was not phosphorylated by purified MAPK, indicating that Thr388 is phosphorylated by MAPK. We then produced and analysed MAPKKs mutated at potential serine phosphorylation sites (S218A-MAPKK and S222A-MAPKK). The wild-type MAPKK (WT-MAPKKK), T388A-MAPKK and a kinase-deficient (K97S)-MAPKK were phosphorylated efficiently by MAPKK-Ks purified from Xenopus eggs, and WT-MAPKK and T388A-MAPKK became activated. In contrast, neither S218A-MAPKK nor S222A-MAPKK was phosphorylated and activated efficiently by the Xenopus MAPKK-Ks. Similarly, WT-MAPKK, but not S218A-MAPKK or S222A-MAPKK, was activated efficiently by an active Raf-1 immunoprecipitate. However, when the recombinant STE11, a putative MAPKK-K in S. cerevisiae, was used as a source of MAPKK-K, S218A-MAPKK as well as WT-MAPKK, but not S222A-MAPKK, was phosphorylated and activated. Furthermore, replacement of Ser222 with an acidic residue (S222E) elevated substantially the basal kinase activity of MAPKK, while replacement of Ser218 (S218E) did not. These results may suggest an essential role for Ser222 phosphorylation in activating Xenopus MAPKK.

Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone-induced MAP kinase activation. Moreover, progesterone-induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos-induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti-MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.

Regulation and function of the MAP kinase cascade in Xenopus oocytes.

In Xenopus oocytes, activation of MAP kinase occurs during meiotic maturation through a protein kinase cascade (the MAP kinase cascade), which is utilized commonly in various intracellular signaling pathways in eukaryotes. Studies with a neutralizing antibody against Xenopus MAP kinase kinase (MAPKK), a direct upstream activator for MAP kinase, have shown that the MAP kinase cascade plays a crucial role in both initiating oocyte maturation and inducing metaphase arrest.

cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates.

A Xenopus 45 kDa protein has been identified as an immediate upstream factor sufficient for full activation of MAP kinase, and is shown to be capable of undergoing autophosphorylation on serine, threonine and tyrosine residues. In this study, we show that purified 45 kDa protein can phosphorylate a kinase-negative mutant of Xenopus MAP kinase on tyrosine and threonine residues, suggesting that the 45 kDa protein functions as a MAP kinase kinase to activate MAP kinase. We then report the cloning and sequencing of a full-length cDNA encoding this 45 kDa MAP kinase kinase, and show that it is highly homologous to four protein kinases in fission and budding yeasts: byr1, wis1, PBS2 and STE7. These yeast kinases are therefore suggested to function as a direct upstream activator for a presumed MAP kinase homolog in each signal transduction pathway involved in the regulation of cell cycle progression or cellular responses to extracellular signals. Finally, we report bacterial expression of recombinant MAP kinase kinase that can be phosphorylated and activated by Xenopus egg extracts.

Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection.

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.