Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation.

Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin ß-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

Structure of msj-1 gene in mice and humans: a possible role in the regulation of male reproduction.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj-1 gene and searched for putative msj-1-like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions (Ha and Hb) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster (ugt1a@). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha. Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

Ab initio identification of putative human transcription factor binding sites by comparative genomics.

BACKGROUND: Understanding transcriptional regulation of gene expression is one of the greatest challenges of modern molecular biology. A central role in this mechanism is played by transcription factors, which typically bind to specific, short DNA sequence motifs usually located in the upstream region of the regulated genes. We discuss here a simple and powerful approach for the ab initio identification of these cis-regulatory motifs. The method we present integrates several elements: human-mouse comparison, statistical analysis of genomic sequences and the concept of coregulation. We apply it to a complete scan of the human genome. RESULTS: By using the catalogue of conserved upstream sequences collected in the CORG database we construct sets of genes sharing the same overrepresented motif (short DNA sequence) in their upstream regions both in human and in mouse. We perform this construction for all possible motifs from 5 to 8 nucleotides in length and then filter the resulting sets looking for two types of evidence of coregulation: first, we analyze the Gene Ontology annotation of the genes in the set, searching for statistically significant common annotations; second, we analyze the expression profiles of the genes in the set as measured by microarray experiments, searching for evidence of coexpression. The sets which pass one or both filters are conjectured to contain a significant fraction of coregulated genes, and the upstream motifs characterizing the sets are thus good candidates to be the binding sites of the TF's involved in such regulation. In this way we find various known motifs and also some new candidate binding sites. CONCLUSION: We have discussed a new integrated algorithm for the "ab initio" identification of transcription factor binding sites in the human genome. The method is based on three ingredients: comparative genomics, overrepresentation, different types of coregulation. The method is applied to a full-scan of the human genome, giving satisfactory results.

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron.

We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.

Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control.

The cyclin-dependent kinase inhibitor p21(Cip1/WAF1) has been implicated as an inducer of differentiation. However, although expression of p21 is increased in postmitotic cells immediately adjacent to the proliferative compartment, its expression is decreased in cells further along the differentiation program. Expression of the p21 protein was decreased in terminally differentiated primary keratinocytes of mice, and this occurred by a proteasome-dependent pathway. Forced expression of p21 in these cells inhibited the expression of markers of terminal differentiation at both the protein and messenger RNA levels. These inhibitory effects on differentiation were not observed with a carboxyl-terminal truncation mutant or with the unrelated cyclin-dependent kinase inhibitor p16(INK4a), although all these molecules exerted similar inhibition of cell growth. These findings reveal an inhibitory role of p21 in the late stages of differentiation that does not result from the effects of p21 on the cell cycle.

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.