Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2kyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2kyclin B, cdk2kyclin A, cdk2lcyclin E and cdkSlp35 only are substantially inhibited (IC,, values of 0.65, 0.7, 0.7 and 0.2 pM, respectively). cdk4kyclin D1 and cdk6kyclin D2 are very poorly inhibited by roscovitine (IC,, > 100 pM). Extracellular regulated kinases erkl and erk2 are inhibited with an IC,, of 34 pM and 14 pM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC,,] of 16 pM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2kyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

Expression of rat TrkA in Xenopus spinal neurons confers responsiveness of these neurons to nerve growth factor (NGF) in assays of neuronal survival and growth cone chemotropism. Mutational analysis indicates that coactivation of phospholipase C-gamma (PLC-gamma) and phosphoinositide 3-kinase (PI3-kinase) by specific cytoplasmic domains of TrkA is essential for triggering chemoattraction of the growth cone in an NGF gradient. Uniform exposure of TrkA-expressing neurons to NGF resulted in a cross-desensitization of turning responses induced by a gradient of netrin-1, brain-derived neurotrophic factor (BDNF), or myelin-associated glycoprotein (MAG) but not by a gradient of collapsin-1/semaphorin III/D or neurotrophin-3 (NT-3). These results, together with the effects of pharmacological inhibitors, support the notion that there are common cytosolic signaling pathways for two separate groups of guidance cues, one of which requires coactivation of PLC-gamma and PI3-kinase pathways.

Neurons have the remarkable ability to polarize even in symmetrical in vitro environments. Although recent studies have shown that asymmetric intracellular signals can induce neuronal polarization, it remains unclear how these polarized signals are organized without asymmetric cues. We describe a novel protein, named shootin1, that became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually, shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, whereas repressing shootin1 expression inhibited polarization. Overexpression and RNA interference data suggest that shootin1 is required for spatially localized phosphoinositide-3-kinase activity. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma; inhibiting this transport prevented its asymmetric accumulation in neurons. We propose that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.

Detailed information on innervation of the histaminergic system in the brain is essential to an understanding of the physiological roles of this system. In a previous immunocytochemical study with antihistidine decarboxylase (HDC) antibody, we detected extensive networks of histaminergic fibers in many areas of the rat brain (Watanabe et al., '84). In the present study, we improved the immunocytochemical procedure and examined the detailed distribution of histaminergic innervation in the rat brain with anti-HDC antibody. As reported previously, the highest concentrations of fibers were found in the hypothalamic nuclei and medial forebrain bundle. With the modified procedure, we detected more dense networks of HDC-immunoreactive (HDCI) fibers. Furthermore, we demonstrated for the first time the existence of HDCI fibers in other regions, namely, the thalamic nuclei, median eminence, fimbria of the hippocampus, habenular nuclei, superior colliculus, nucleus of the optic tract, parabrachial nuclei, mesencephalic trigeminal nucleus, superior, lateral, and spinal vestibular nuclei, posterior lobe of the hypophysis, and vascular organ of the lamina terminalis. We also found dense transverse fibers in the retrochiasmatic area and supraoptic decussation, which suggests bilateral innervation of the histaminergic system. These results indicate that innervation of the rat brain by the histaminergic system is more extensive than observed previously.

No abstract

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.