Using the yeast two hybrid system and overlay assays, a putative rho/rac effector, citron, has been identified that interacts with the GTP-bound forms of rho and rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C6H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rho/rac binding site. Others putative rho effectors characterized by a common rho binding motif have been identified. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners (Madaule, 1995).
A novel serine/threonine kinase belonging to the myotonic dystrophy kinase family has been identified. The kinase can be produced in at least two different isoforms: (1) an 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; (2) an approximately 54-kDa protein [CRIK-short kinase (SK)], that 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 constitutively 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 (Di Cunto, 1988).
Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in the regulation of cytokinesis. To further investigate in vivo its biologic functions, Citron-K gene has been inactivated 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. These 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 (Di Cunto, 2000).
Cytokinesis is an essential step in neurogenesis, yet the mechanisms that control cytokinesis in the developing CNS are not well understood. The flathead (fh) mutation in rat results in cytokinesis failure in neural progenitors followed by apoptosis and a dramatic reduction in CNS growth. Evidence is presented that the fh mutation is caused by a single base deletion in exon 1 of the gene encoding Citron-Kinase (Citron-K). This base deletion causes a premature stop codon at the 27th codon in the N-terminal kinase domain of Citron-K, and Western blot and immunocytochemical analysis show that the Citron-K protein is absent in proliferative zones in fh/fh mutant embryos. Citron-K protein is normally expressed along the ventricular zone (VZ) surface and localizes to cleavage furrows of both symmetrically and asymmetrically dividing progenitors. In addition, Citron-K colocalizes with RhoA at cleavage furrows in wild-type (wt) embryos, whereas RhoA expression is reduced at the VZ surface and is absent from many cytokinesis furrows in homozygous fh/fh mutants. These results, together with evidence from a recently described induced mutation in mice, indicate that the flathead mutation is in the Citron-K gene and that Citron-K may act with RhoA to ensure the progression of cytokinesis in neuronal progenitors (Sarkisian, 2002).
Citron Kinase (Citron-K) is a cell cycle-dependent protein regulating the G(2)/M transition in hepatocytes. Synchronization studies demonstrate that expression of the Citron-K protein starts at the late S and/or the early G(2) phase after that of cyclin B1. Expression of Citron-K is developmentally regulated. Levels of Citron-K mRNA and protein are highest in embryonic liver and gradually decrease after birth. Citron-K exists in interphase nuclei and begins to disperse into the cytoplasm at prophase. It concentrates at the cleavage furrow and midbody during anaphase, telophase, and cytokinesis, implicating a role in the control of cytokinesis. However, studies with knockouts show that Citron-K is not essential for cytokinesis in hepatocytes. Instead, loss of Citron-K causes a significant increase of G(2) tetraploid nuclei in one-week-old rat and mouse liver. In addition, Citron-K deficiency triggers apoptosis in a small subset of embryonic liver cells. In summary, these data demonstrate that Citron-K has a distinct cell cycle-dependent expression pattern and cellular localization as a downstream target of Rho-GTPase and functions in the control of G(2)/M transition in the hepatocyte cell cycle (Liu, 2003).
Small GTPases of the rho family regulate the extensive rearrangements of the cytoskeleton that characterize neuronal differentiation. Citron kinase is a target molecule for activated rhoA, previously implicated in control of cytokinesis. In addition, Citron kinase plays an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, it has been shown that citron kinase is involved in the morphologic differentiation of N1E-115 neuroblastoma cells induced by serum starvation. More importantly, quantitative analysis of citron kinase knockout cerebral cortex shows that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons (Di Cunto, 2003).
During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. A splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. Overexpression of citron mutants results in the production of multinucleate cells and a kinase-active mutant causes abnormal contraction during cytokinesis. It is proposed that citron kinase regulates cytokinesis at a step after Rho in the contractile process (Madaule, 1998).
Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, Citron-K localization in cell cycle was monitored. Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase. Rho colocalizes with Citron-K in the cortex of anaphase to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody. Inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevents its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, active Val14RhoA was expressed in interphase cells expressing GFP-Citron-K. Val14RhoA expression transfers Citron-K to the ventral cortex of interphase cells, where it forms band-like structures in a complex with Rho. This structure localizes at the same plane as actin stress fibers, and they exclude one another. Disruption of F-actin abolishes the band and disperses the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in the cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells (Eda, 2001).
During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis reveals that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. These data indicate that Citron kinase is specifically required for cytokinesis of the male germ line (Di Cunto, 2002).
Successful cell division in neural progenitors in the neocortical ventricular zone (VZ), as in all dividing cells, depends critically upon coordinating chromosome segregation during mitosis with cytokinesis. This coordination further suggests that common molecular regulators may link events in mitosis with those in cytokinesis. Recent genetic evidence indicates that cytokinesis in CNS neuronal progenitors, but not in most other cell types of the body, requires the function of citron kinase. In neocortex, citron kinase is most critical for neurogenic cytokinesis. In citron kinase null mutants, a large proportion of neuronal cells within neocortex are binucleate; however, very few glial cells are binucleate. In addition, confocal time-lapse imaging of mitoses at the VZ surface shows that citron kinase is also necessary for phases of the cell cycle just prior to cytokinesis. Deficits in mitosis seen in mutants indicate aberrant mitotic spindle function, and like deficits in cytokinesis, these occur in some but not all cells at the VZ surface. Citron kinase is therefore an essential multifunctional regulator of cell divisions in the VZ, and may serve to coordinate chromosome segregation with cytokinesis in neuronal precursors (LoTurco, 2003).
Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. Citron kinase has been shown to phosphorylate regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase does not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. The expression of the kinase domain of citron kinase results in an increase in MLC di-phosphorylation. Furthermore, the kinase domain is able to increase di-phosphorylation and restore stress fiber assembly even when ROCK is inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increases di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis (Yamashiro, 2003).
The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. The brain-specific Rho-binding protein Citron-N is shown to be enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, these results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics (Camera, 2003).
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