Expression of stathmin and SCG10 proteins in the olfactory neurogenesis during development and after lesion in the adulthood.

Stathmin and SCG10 belong to a family of phosphoproteins associated to cell proliferation and differentiation. In the present study, we have analyzed immunocytochemically the distribution of these proteins during neurogenesis in the mouse olfactory system, from midgestation to adulthood. Data show that already at embryonic day 12, stathmin and SCG10 immunoreactivities were present in the olfactory and vomeronasal neurons, and their number increased greatly, colocalizing with neuronal specific tubulin, a marker of immature neurons. Later on up to adulthood, the distribution of stathmin and SCG10 became progressively restricted to a few immature receptor and chemosensory neurons. Significantly, in the olfactory epithelium, stathmin was seen in immature neurons and also in basal cells representing precursors of neuronal elements. Interestingly, before birth stathmin and SCG10 immunopositive cells were seen outside the olfactory epithelium, seemingly migrating toward the olfactory bulb. After regeneration in the adult following peripheral lesion of the olfactory epithelium, stathmin and SCG10 were again strongly expressed and generally colocalized with neuronal specific tubulin immunoreactivity. Overall these results indicate that stathmin and SCG10 are expressed in immature olfactory neurons as well as in the migrating cells generated from the olfactory epithelium, supporting the role of these proteins in neurogenesis and cell migration.

The stathmin family -- molecular and biological characterization of novel mammalian proteins expressed in the nervous system.

Stathmin is a ubiquitous phosphoprotein proposed to be a relay integrating various intracellular signaling pathways. Its high phylogenetic conservation and the identification of the related molecules, SCG10 in rat and XB3 in Xenopus, suggested the existence of a stathmin-related family. A systematic PCR-based approach allowed the identification of several novel mammalian sequences of which two coded for expressed members of the stathmin family; the translated RB3 sequence shares 88% amino-acid identity with that of XB3 and is thus its rat homologue, and RB3' corresponds to an alternatively spliced product of the same gene, encoding a truncated form. Within their stathmin-like domain, the alpha helix, probably responsible for coiled-coil protein-protein interactions, is conserved, as well as are two consensus phosphorylation sites; in their N-terminal extension domain, two cystein residues most likely responsible for membrane attachment through palmitoylation, are present in RB3/RB3' as in SCG10. The novel identification and characterization of the corresponding proteins showed that all three are associated with the particulate, membrane-containing fraction. They furthermore display several spots of decreasing pI on two-dimensional immunoblots, suggesting that they are phosphorylated in vivo. As for SCG10, RB3 mRNA is detectable only in the nervous system by in situ hybridization, but at similar levels in the newborn and the adult brain as revealed by Northern blots, whereas SCG10 expression decreases in the adult. Furthermore, RB3 mRNA is undetectable in PC12 cells, whereas SCG10 mRNA increases after treatment with nerve growth factor, inducing neuronal differentiation. In conclusion, we demonstrate here the existence of a highly conserved stathmin-related family in mammals, of which each member seems to play specific roles, related to the control of cell proliferation and activities for stathmin and to that of neuronal differentiation for SCG10, the novel RB3/RB3' proteins being rather related to the expression of differentiated neuronal functions.

The Ulip family phosphoproteins--common and specific properties.

The search for intracellular phosphoproteins implicated in the regulation of neuronal differentiation led to the identification of Ulip1, a mammalian protein related to the Caenorhabditis elegans unc-33 gene product [Byk, T., Dobransky, T., Cifuentes-Diaz, C. & Sobel, A. (1996) J. Neurosc. 16, 688-701]. The expression level and phosphorylation pattern of Ulip1 were shown to be strongly regulated during development and neuronal differentiation. We have isolated three additional complete coding sequences for members of the Ulip family in the mouse, Ulips 2-4, all preferentially expressed in the nervous system. Furthermore, two Ulip sequences, Ulips A and Ulips B, could be identified in C. elegans. The Ulip family is highly conserved throughout evolution (more than 96 % for Ulips 1-3 and 92.5 % for Ulip4 between mouse and human) and the various members of the family within a single species display about 75% similarity. Sequence comparisons further reveal several highly similar domains and subdomains, including a 32-amino-acid region highly conserved from a bacterial hydantoinase to human Ulips. Two-dimensional immunoblot analysis of in vitro translated Ulips 1-4 demonstrates the existence, for each Ulip protein, of several, most probably differentially phosphorylated forms, in agreement with the presence of conserved phosphorylation consensus sites within their sequences. The expression of Ulips 1-4 mRNAs is differentially regulated during development and nerve-growth-factor-induced neuronal differentiation of PC12 cells. Our results indicate a differential, possibly complementary role of phosphoproteins of the highly conserved Ulip family in the control of neuronal differentiation, in relation with the development and plasticity of the nervous system.

SCLIP: a novel SCG10-like protein of the stathmin family expressed in the nervous system.

Stathmin is a cytosolic phosphoprotein previously described as a ubiquitous relay integrating various signaling pathways. It is the generic element of a protein family in mammals as in Xenopus, including SCG10, a neuron-specific, growth-associated protein, and RB3/XB3, related to the expression of differentiated functions of mature cells of the nervous system. In an extensive search for other members of the stathmin family, we identified cDNAs coding for two novel stathmin-related proteins: (a) a cDNA from a rat striatum cDNA library codes for RB3", a splice variant of RB3, with an additional basic domain in its N-terminal region; and (b) another cDNA identified through a systematic search in EST databases codes for a novel protein, SCLIP, for "SCG10-like protein," displaying 70% identity with SCG10 and sharing the same domain organization, with an N-terminal domain likely involved in membrane attachment and a C-terminal stathmin-like domain. Northern blot analysis as well as in situ hybridization on 14-day rat embryos showed that SCLIP mRNA is expressed only in neural structures. SCLIP mRNA is expressed at comparable levels in neonatal and adult rat brain, suggesting a potential role not only in the acquisition, but also in the expression of differentiated neuronal functions.

Differential, regional, and cellular expression of the stathmin family transcripts in the adult rat brain.

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and previously described as a relay integrating diverse intracellular signaling pathways. Stathmin is the generic element of a mammalian protein family including SCG10, SCLIP, and RB3 with its splice variants RB3' and RB3". In contrast with stathmin, SCG10, SCLIP, and RB3/RB3'/RB3" are exclusively expressed in the nervous system, stathmin and SCG10 being mostly expressed during cell proliferation and differentiation, and SCLIP and RB3 rather in mature neural cells. To further understand their specific roles in the CNS, we compared the localization of the stathmin, SCG10, SCLIP, and RB3 transcripts in adult rat brain. Northern blot analysis as well as in situ hybridization experiments showed that all stathmin-related mRNAs are expressed in a wide range of adult rat brain areas. At a regional level, SCG10 and SCLIP appear generally distributed similarly except in a few areas. The pattern of expression of the RB3 transcript is very different from that of the three other members of the stathmin family. Furthermore, unlike SCG10 and SCLIP, which were detected only in neurons, but like stathmin, RB3 was detected in neurons and also in glial cells of the white matter. Altogether, our results suggest distinct roles for each member of the stathmin-related phosphoprotein family, in regard to their specific regional and cellular localization in the rat brain.

Stathmin family proteins display specific molecular and tubulin binding properties.

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3'/RB3") are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific "stathmin-like domain" and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two alpha/beta tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3'. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.

KIS is a protein kinase with an RNA recognition motif.

Protein phosphorylation is involved at multiple steps of RNA processing and in the regulation of protein expression. We present here the first identification of a serine/threonine kinase that possesses an RNP-type RNA recognition motif: KIS. We originally isolated KIS in a two-hybrid screen through its interaction with stathmin, a small phosphoprotein proposed to play a general role in the relay and integration of diverse intracellular signaling pathways. Determination of the primary sequence of KIS shows that it is formed by the juxtaposition of a kinase core with little homology to known kinases and a C-terminal domain that contains a characteristic RNA recognition motif with an intriguing homology to the C-terminal motif of the splicing factor U2AF. KIS produced in bacteria has an autophosphorylating activity and phosphorylates stathmin on serine residues. It also phosphorylates in vitro other classical substrates such as myelin basic protein and synapsin but not histones that inhibit its autophosphorylating activity. Immunofluorescence and biochemical analyses indicate that KIS overexpressed in HEK293 fibroblastic cells is partly targetted to the nucleus. Altogether, these results suggest the implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors.

The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network.

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3' and RB3". Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3" were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).

Stathmin and its phosphoprotein family: general properties, biochemical and functional interaction with tubulin.

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.