Centrosome-associated NDR kinase regulates centrosome duplication

Human NDR kinases are upregulated in some cancer types, yet their functions still remain undefined. Here, we report the first known function of a mammalian NDR kinase by demonstrating that human NDR directly contributes to centrosome duplication. A subpopulation of endogenous NDR localizes to centrosomes in a cell-cycle-dependent manner. Overexpression of NDR resulted in centrosome overduplication in a kinase-activity-dependent manner, while expression of kinase-dead NDR or depletion of NDR by small interfering RNA (siRNA) negatively affected centrosome duplication. By targeting NDR to the centrosome, we show that the centrosomal pool of NDR is sufficient to generate supernumerary centrosomes. Furthermore, our data indicate that NDR-driven centrosome duplication requires Cdk2 activity and that Cdk2-induced centrosome amplification is affected upon reduction of NDR activity. Overall, considering that centrosome overduplication is linked to cellular transformation, our observations may also provide a molecular link between mammalian NDR kinases and cancer.     

Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication

Background information. Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over‐duplication in addition to the re‐replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal‐localized proteins is still unidentified. Results. We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell‐cycle dependent and mediated by the actin‐related protein, Arp1, one subunit of the dynein—dynactin complex. Disruption of the integrity of the dynein—dynactin complex by overexpression of dynamitin/p50, a well‐characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP‐inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled‐coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA‐MB‐231 cells led to centrosome over‐duplication. Conversely, overexpression of geminin inhibits centrosome over‐duplication induced by HU in S‐phase‐arrested cells, and the coiled‐coil‐motif‐mediated centrosomal localization of geminin is required for its inhibition of centrosome over‐duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication. Conclusions. The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1‐mediated and dynein—dynactin‐dependent. The coiled‐coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.     

Mammalian protein histidine kinases

The existence of protein kinases, known as histidine kinases, which phosphorylate their substrates on histidine residues has been well documented in bacteria and also in lower eukaryotes such as yeast and plants. Their biological roles in cellular signalling pathways within these organisms have also been well characterised. The evidence for the existence of such enzymes in mammalian cells is much less well established and little has been determined about their cellular functions. The aim of the current review is to present a summary of what is known about mammalian histidine kinases. In addition, by consideration of the chemistry of phosphohistidine, what is currently known of some mammalian histidine kinases and the way in which they act in bacteria and other eukaryotes, a general role for mammalian histidine kinases is proposed. A histidine kinase phosphorylates a substrate protein, by virtue of the relatively high free energy of hydrolysis of phosphohistidine the phosphate group is easily transferred to either a small molecule or another protein with which the phosphorylated substrate protein specifically interacts. This allows a signalling process to occur, which may be downregulated by the action of phosphatases. Given the known importance of protein phosphorylation to the regulation of almost all aspects of cellular function, the investigation of the largely unexplored area of histidine phosphorylation in mammalian cells is likely to provide a greater understanding of cellular action and possibly provide a new set of therapeutic drug targets.     

NDR kinases regulate essential cell processes from yeast to humans

Members of the NDR (nuclear Dbf2-related) protein-kinase family are essential components of pathways that control important cellular processes, such as morphological changes, mitotic exit, cytokinesis, cell proliferation and apoptosis. Recent progress has shed light on the mechanisms that underlie the regulation and function of the NDR family members. Combined data from yeast, worms, flies, mice and human cells now highlight the conserved and important roles of the different NDR kinases in distinct cellular processes.     

cGMP-dependent protein kinases: properties, regulation and biological role

The data available in literature on properties, methods of regulation and biological role of cGMP-dependent protein kinases are analyzed and generalized. Evidence on distribution of these enzymes in different tissues and intracellular compartments is presented. Physicochemical properties of cGMP-dependent protein kinases from different sources are analyzed. Peculiarities of their primary structure, regulation of their activity at the expense of autophosphorylation as well as in the presence of the modulator protein, metal ions and other factors are considered. These data permit postulating importance of cGMP-dependent phosphorylation in some physiological and biochemical processes.     

A role for PML3 in centrosome duplication and genome stability

The promyelocytic leukemia gene (PML), which is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL), encodes a multifunctional protein involved in several important cellular functions. Herein, we demonstrate that PML is localized to centrosomes and that PML deficiency leads to centrosome amplification. By using PML isoform-specific antibodies, we found PML3-specific association with the centrosome and the pole of the mitotic spindle. PML3 deficiency leads to dysregulation of the centrosome duplication checkpoint. Furthermore, PML3 physically interacts with Aurora A and regulates its kinase activity. Specific knockdown of PML3 activates Cdk2/cyclin kinase activity. The results of this study implicate a direct role for PML3 in the control of centrosome duplication through suppression of Aurora A activation to prevent centrosome reduplication.     

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca²⁺/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation. shear stress; nitric oxide; endothelial cells; protein kinases     

Mitogen-activated protein kinases and their role in regulation of cellular processes

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes connecting cell-surface receptors to critical regulatory targets within cells. The three major MAPK cascades are known, the extracellular signal-regulated protein kinase (ERK) cascade, c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and p38-MAPK cascade. This paper is focused on characterization of these MAPK cascades in terms of their distribution and biological role in some pathological processes (apoptosis, hypertrophy) with a special orientation on the role of MAPKs in cardiovascular system during ischemia/reperfusion.     

The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.     

Duplicating dangerously: linking centrosome duplication and aneuploidy

Centrosomes are far more fascinating than the first explorers of this organelle a century ago could ever have imagined. Recent evidence indicates that deregulation of centrosome duplication affects centrosome number and promotes aneuploidy, features characteristic of human tumors.     

Redox regulation of protein kinases

Abstract

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous hydrogen peroxide (H₂O₂) by membrane-bound NADPH oxidases. In turn, H₂O₂ can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H₂O₂ regarding kinase activity, as well as the components involved in H₂O₂ production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H₂O₂ through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiological and pathological H₂O₂ responses.     

The role of protein phosphatases in the regulation of mitogen and stress-activated protein kinases.

It is now established that a family of dual-specificity protein phosphatases are able to interact with mitogen and stress-activated protein kinases in a highly specific manner to differentially regulate these enzymes in mammalian cells. A role for these proteins in negative feedback regulation of MAP kinase activity is also supported by genetic and biochemical studies in yeasts and Drosophila. More recently it has become clear that other classes of protein phosphatase also play key roles in the regulated dephosphorylation of MAP kinases, including tyrosine-specific protein phosphatases and serine/threonine protein phosphatases. It is likely that a complex balance between upstream activators and these different classes of MAP kinase specific phosphatase are responsible for determining, at least in part, the magnitude and duration of MAP kinase activation and hence the physiological outcome of signalling.     

The role of protein kinases in the regulation of plant growth and development

We review the role of protein kinases in plant hormone-mediatedsignalling, nutrient signalling and cell cycle control and in the crosstalkbetween these different contributors to plant growth regulation. The areas ofhormone-mediated signalling covered include ABA-mediated responses to osmoticstress, wounding and pathogen attack, as well as ethylene and cytokininsignalling pathways. These areas involve members of several major protein kinasefamilies, including the SNFl-related protein kinase-2 (SnRK2) subfamily, thecalcium-dependent protein kinase (CDPK) family, the mitogen activated protein(MAP) kinase family, the glycogen synthase kinase (GSK)- 3/shaggy family and thereceptor-like protein kinase (RPK) family. In the section on nutrient signallingwe review the role of SnRK1 protein kinases in the global regulation of carbonmetabolism, including aspects of sugar sensing and assimilate partitioning, andwhat is known about nitrogen and sulphur nutrient signalling. In the cell cyclesection, we summarise progress in the elucidation of cell cycle control systemsin plants and discuss the interaction between cell cycle control anddevelopment. We expand further on the hypothesis of crosstalk between differentsignalling pathways in a separate section in which we discuss evidence forinteraction between plant growth regulators and the cell cycle, betweendifferent nutrient signalling pathways, between nutrient and cell cyclesignalling and between nutrient and ABA signalling.     

Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling

Interactions between adhesion molecules, agglutinins, on the surfaces of the flagella of mt+ and mt- gametes in Chlamydomonas rapidly generate a sexual signal, mediated by cAMP, that prepares the cells for fusion to form a zygote. The mechanism that couples agglutinin interactions to increased cellular levels of cAMP is unknown. In previous studies on the adenylyl cyclase in flagella of a single mating type (i.e., non-adhering flagella) we presented evidence that the gametic form of the enzyme, but not the vegetative form, was regulated by phosphorylation and dephosphorylation (Zhang, Y., E. M. Ross, and W. J. Snell. 1991. J. Biol. Chem. 266:22954-22959; Zhang, Y., and W. J. Snell. 1993. J. Biol. Chem. 268:1786-1791). In the present report we describe studies on regulation of flagellar adenylyl cyclase during adhesion in a cell-free system. The results show that the activity of gametic flagellar adenylyl cyclase is regulated by adhesion in vitro between flagella isolated from mt+ and mt- gametes. After mixing mt+ and mt- flagella together for 15 s in vitro, adenylyl cyclase activity was increased two- to threefold compared to that of the non-mixed (non- adhering), control flagella. This indicates that the regulation of gametic flagellar adenylyl cyclase during the early steps of fertilization is not mediated by signals from the cell body, but is a direct and primary response to interactions between mt+ and mt- agglutinins. By use of this in vitro assay, we discovered that 50 nM staurosporine (a protein kinase inhibitor) blocked adhesion-induced activation of adenylyl cyclase in vitro, while it had no effect on adenylyl cyclase activity of non-adhering gametic flagella. This same low concentration of staurosporine also inhibited adhesion-induced increases in vivo in cellular cAMP and blocked subsequent cellular responses to adhesion. Taken together, our results indicate that flagellar adenylyl cyclase in Chlamydomonas gametes is coupled to interactions between mt+ and mt- agglutinins by a staurosporine- sensitive activity, probably a protein kinase.     

Exon duplication from a fork head to a homeodomain protein

The evolution of complex organisms such as animals requires a large expansion of the number of genes controlling developmental events. In addition, it is thought that domains are shuffled between genes to further increase the complexity and generate new types of genes and functions. Working with the Caenorhabditis elegans homeobox gene ceh-43, the orthologue of fly Distal-less (Dll), we observed sequence similarity to the C. elegans gene fkh-1. Now, with the complete genomic sequence available, we examined this similarity in detail. The region of similarity is confined essentially to one exon in the carboxy terminus of the two genes. Based on the gene structure, we think that an exon of fkh-1 was duplicated to the carboxy terminus of ceh-43, where it was incorporated as the last exon. This duplication event seems to have happened recently since the similarity on the nucleotide level is higher than the sequence similarity between fkh-1 of C. elegans and C. briggsae. Potentially the duplication event was mediated via a short region of sequence similarity between the two open reading frames of the genes. This duplication event clear shows that a part of a gene can successfully be juxtaposed to another gene. These events may perhaps not be rare. Received: 28 March 1999 / Accepted: 8 June 1999     

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr¹⁹⁹ by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr¹⁹⁹ remains at centrosomes. It has been shown that Thr¹⁹⁹ phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr¹⁹⁹. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr¹⁹⁹-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.     

A novel role for the deubiquitinase USP1 in the control of centrosome duplication

Defects in the regulation of centrosome duplication lead to tumorigenesis through abnormal cell division and resulting chromosome missegregation. Therefore, maintenance of accurate centrosome number is critical for cell fate. The deubiquitinating enzyme USP1 plays important roles in DNA repair and cell differentiation. Importantly, increased levels of USP1 are detected in certain types of human cancer, but little is known about the significance of USP1 overexpression in cancer development. Here we show that Usp1 plays a novel role in regulating centrosome duplication. The ectopic expression of wild-type Usp1, but not C90S Usp1 (catalytically inactive mutant form), induced centrosome amplification. Conversely, ablation of Usp1 in mouse embryonic fibroblasts (MEFs) showed a significant delay in centrosome duplication. Moreover, Usp1-induced centrosome amplification caused abnormal mitotic spindles, chromosome missegregation and aneuploidy. Interestingly, loss of inhibitor of DNA binding protein 1 (ID1) suppressed Usp1-induced centrosome amplification. Taken together, our results strongly suggest that Usp1 is involved in the regulation of centrosome duplication, at least in part via ID1, and Usp1 may exert its oncogenic activity, partially through inducing centrosome abnormality.