Citron Kinase Research Articles

Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission

The guanine nucleotide-exchange factor (GEF) Ect2 is essential for cytokinesis. Here we studied the subcellular localization of Ect2 and examined the consequences of either depleting or overexpressing Ect2 in human cells. We show that in mitotic cells Ect2 localizes to the central spindle and to the cell cortex. The latter association is mediated through a PH domain in Ect2 and central spindle localization requires the MKlp1-MgcRacGAP and MKlp2-Aurora-B complexes. Ect2 directly interacts with MKlp1-MgcRacGAP through its BRCT domain, whereas MKlp2-Aurora-B probably exerts a regulatory role in Ect2 central spindle targeting. Depletion of Ect2 impaired cleavage furrow formation and RhoA and Citron kinase failed to accumulate at the cleavage furrow. Ect2 displacement from the central spindle revealed that physiological levels of this protein in this location are not crucial for RhoA activation and cytokinesis. In cells overexpressing appropriate N-terminal Ect2 fragments, RhoA and Citron kinase localized to the cleavage furrow and ingression occurred, but abscission failed. This failure could be correlated with the persistence of these fragments at structures surrounding the midbody, suggesting that abscission requires the displacement of Ect2 from the contractile ring and its re-import into the nucleus.

KIF14 and citron kinase act together to promote efficient cytokinesis

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735–1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877–885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175–1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711–5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832–1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237–3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115–127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.

Citron Kinase, a RhoA Effector, Enhances HIV-1 Virion Production by Modulating Exocytosis

Citron Kinase, a RhoA Effector, Enhances HIV-1 Virion Production by Modulating Exocytosis

The Structure of Dimeric ROCK I Reveals the Mechanism for Ligand Selectivity

ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation.

Efficient Protein Trafficking Requires Trailer Hitch, a Component of a Ribonucleoprotein Complex Localized to the ER in Drosophila

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. We have identified Drosophila trailer hitch (tral) as a gene that is required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral revealed that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell.

Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression

Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal.

PAK and other Rho-associated kinases--effectors with surprisingly diverse mechanisms of regulation.

The Rho GTPases are a family of molecular switches that are critical regulators of signal transduction pathways in eukaryotic cells. They are known principally for their role in regulating the cytoskeleton, and do so by recruiting a variety of downstream effector proteins. Kinases form an important class of Rho effector, and part of the biological complexity brought about by switching on a single GTPase results from downstream phosphorylation cascades. Here we focus on our current understanding of the way in which different Rho-associated serine/threonine kinases, denoted PAK (p21-activated kinase), MLK (mixed-lineage kinase), ROK (Rho-kinase), MRCK (myotonin-related Cdc42-binding kinase), CRIK (citron kinase) and PKN (protein kinase novel), interact with and are regulated by their partner GTPases. All of these kinases have in common an ability to dimerize, and in most cases interact with a variety of other proteins that are important for their function. A diversity of known structures underpin the Rho GTPase-kinase interaction, but only in the case of PAK do we have a good molecular understanding of kinase regulation. The ability of Rho GTPases to co-ordinate spatial and temporal phosphorylation events explains in part their prominent role in eukaryotic cell biology.

Drosophila citron kinase is required for the final steps of cytokinesis.

The mechanisms underlying completion of cytokinesis are still poorly understood. Here, we show that the Drosophila orthologue of mammalian Citron kinases is essential for the final events of the cytokinetic process. Flies bearing mutations in the Drosophila citron kinase (dck) gene were defective in both neuroblast and spermatocyte cytokinesis. In both cell types, early cytokinetic events such as central spindle assembly and contractile ring formation were completely normal. Moreover, cytokinetic rings constricted normally, leading to complete furrow ingression. However late telophases of both cell types displayed persistent midbodies associated with disorganized F actin and anillin structures. Similar defects were observed in dck RNA interference (RNAi) telophases, which, in addition to abnormal F actin and anillin rings, also displayed aberrant membrane protrusions at the cleavage site. Together, these results indicate that mutations in the dck gene result in morphologically abnormal intercellular bridges and in delayed resolution of these structures, suggesting that the wild-type function of dck is required for abscission at the end of cytokinesis. The phenotype of Dck-depleted cells is different from those observed in most Drosophila cytokinesis mutants but extraordinarily similar to that caused by anillin RNAi, suggesting that Dck and anillin are in the same pathway for completion of cytokinesis.

Expression of the Human Myotonic Dystrophy Kinase-related Cdc42-binding Kinase γ Is Regulated by Promoter DNA Methylation and Sp1 Binding

Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCKalpha and -beta, have been previously identified and characterized. We now describe a novel isoform, MRCKgamma, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCKalpha and -beta, which are expressed ubiquitously, MRCKgamma mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCKgamma showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCKgamma promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCKgamma. Analysis of the MRCKgamma promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCKgamma expression.

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac.

The contractile ring is a highly dynamic structure, but how this dynamism is accomplished remains unclear. Here, we report the identification and analysis of a novel Drosophila gene, sticky (sti), essential for cytokinesis in all fly proliferating tissues. sti encodes the Drosophila orthologue of the mammalian Citron kinase. RNA interference–mediated silencing of sti in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such STICKY-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes suggesting that these GTPases antagonistically regulate STICKY functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. We discuss that antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis.

Citron kinase is a regulator of mitosis and neurogenic cytokinesis in the neocortical ventricular zone.

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, 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.

Role of citron kinase in dendritic morphogenesis of cortical neurons

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. We have found that, in addition, it could play an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, we showed 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 displayed that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons.

Citron kinase, a Rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin II.

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. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased 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.

Citron Kinase Is a Cell Cycle-dependent, Nuclear Protein Required for G2/M Transition of Hepatocytes

Citron Kinase (Citron-K) is a cell cycle-dependent protein regulating the G(2)/M transition in hepatocytes. Synchronization studies demonstrated 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, our 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.

Citron Kinase Is a Cell Cycle-dependent, Nuclear Protein Required for G 2 /M Transition of Hepatocytes

Citron Kinase Is a Cell Cycle-dependent, Nuclear Protein Required for G 2 /M Transition of Hepatocytes

Essential role of citron kinase in cytokinesis of spermatogenic precursors.

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. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, 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 revealed 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. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.

Role of myosin light chain phosphorylation in the regulation of cytokinesis.

Phosphorylation of regulatory light chain (RMLC) of myosin II at Ser19/Thr18 is likely to play important roles in controlling the morphological changes seen during cell division of cultured mammalian cells. Phosphorylation of RMLC regulates the activity of myosin II, an essntial motor for cytokinesis, and phosphorylation of RMLC shows dramatic changes during mitosis. Two exzymes, myosin phosphatase and kinase, control phosphorvlation of RMLC. Myosin phosphatase is activated during mitosis, apparently as a result of mitosis-specific phosphorylation of the myosin phosphatase targeting subunit (MYPT). This activation of myosin phosphatase is likely to result in RMLC dephosphorylation, causing the disassemly of stress fibers and focal adhesions during prophase. The phosphorylation of MYPT is lost in cyotokinesis, which would decrease myosin phosphatase activity. At the same time, ROCK (Rho-kinase) probably phosphorylates MYPT at its inhibitory sites, further decreasing the activity of myosin phosphatase. These changes in MYPT phosphorylation would raise RMLC phosphorylation, leading to the activation of myosin II for cyotokinesis. RMLC phosphorylation is also regulated by several RMLC kinases including ROCK (Rho-kinase), MLCK and citron kinase, all of which are localized at cleavage furrows. Future studies should examine whether these multiple kinases are redundant or whether they control distinct aspects of cell division.

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.

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.

Role of citron kinase as a target of the small GTPase Rho in cytokinesis.

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. We now show that 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. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.