Function and regulation of Aurora／Ipllp kinase family in cell division

During mitosis,the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation,a complex movements orchestrated by mitotic kinases and its effector proteins.Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell.Defects in these processes can lead to aneuploidy or polyploidy.Aurora/Ipllp family, a class of conserved serine/threonine kinases,plays key roles in chromosome segregation and cytokinesis.This article highlights the function and regulation of Aurora/Ipllp family in mitosis and provides potential links between aberrant regulation of Aurora/Ipllp kinases and pathogenesis of human cancer.     

Regulation of Fission Yeast Myosin-II Function and Contractile Ring Dynamics by Regulatory Light-Chain and Heavy-Chain Phosphorylation

We investigated the role of regulatory light-chain (Rlc1p) and heavy-chain phosphorylation in controlling fission yeast myosin-II (Myo2p) motor activity and function during cytokinesis. Phosphorylation of Rlc1p leads to a fourfold increase in Myo2p''s in vitro motility rate, which ensures effective contractile ring constriction and function. Surprisingly, unlike with smooth muscle and nonmuscle myosin-II, RLC phosphorylation does not influence the actin-activated ATPase activity of Myo2p. A truncated form of Rlc1p lacking its extended N-terminal regulatory region (including phosphorylation sites) supported maximal Myo2p in vitro motility rates and normal contractile ring function. Thus, the unphosphorylated N-terminal extension of Rlc1p can uncouple the ATPase and motility activities of Myo2p. We confirmed the identity of one out of two putative heavy-chain phosphorylation sites previously reported to control Myo2p function and cytokinesis. Although in vitro studies indicated that phosphorylation at Ser-1444 is not needed for Myo2p motor activity, phosphorylation at this site promotes the initiation of contractile ring constriction.     

Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.     

Disruption of the autism-related gene Pak1 causes stereocilia disorganization,hair cell loss,and deafness in mice

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases(PAKs)are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout(Pak1à/à) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell(HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of b II-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1à/àmice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.     

p21-activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells

Stimulation of p21-activated kinase-1 (Pak1) signaling promotes motility, invasiveness, anchorage-independent growth and abnormal mitotic assembly in human breast cancer cells. Here, we provide new evidence that, before the onset of mitosis, activated Pak1 is specifically localized with the chromosomes during prophase and on the centrosomes in metaphase and moves to the contraction ring during cytokinesis. To identify mitosis-specific substrates of Pak1, we screened a synchronized G₂–M expression library by using a glutathione transferase Pak1 solid-phase-based kinase reaction. This analysis identified histone H3 as a substrate of Pak1 both in vitro and in vivo, and it specifically interacted with Pak1 but not Pak2 or Pak3. Site-directed mutagenesis indicated that Pak1 phosphorylates histone H3 on Ser10. Expressions of the wild-type, or catalytically active, Pak1 caused it to appear at the poles corresponding to mitotic centrosomes in a variety of mammalian cells. Together, these results suggest for the first time that Pak1 interacts with and phosphorylates histone H3 and may thus influence the Pak1–histone H3 pathway, which in turn may influence mitotic events in breast cancer cells.     

Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function

The F-actin based motor protein myosin II has a key role in cytokinesis. Here we show that the Schizosaccharomyces pombe regulatory light chain (RLC) protein Rlc1p binds to Myo2p in manner that is dependent on the IQ sequence motif (the RLC-binding site), and that Rlc1p is a component of the actomyosin ring. Rlc1p is important for cytokinesis at all growth temperatures and is essential for this process at lower temperatures. Interestingly, all deleterious phenotypes associated with the loss of Rlc1p function are suppressed by deletion of the RLC binding site on Myo2p. We conclude that the sole essential function of RLCs in fission yeast is to relieve the auto-inhibition of myosin II function, which is mediated by the RLC-binding site, on the myosin heavy chain (MHC).     

Roles of Pdk1p, a fission yeast protein related to phosphoinositide-dependent protein kinase, in the regulation of mitosis and cytokinesis

Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network.     

UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.     

Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring. One of the key components of this ring is the F-actin based motor protein myosin II. The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p. Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p. In this study, we have created and characterized S. pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains. Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function. Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities. Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished. Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis. Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p. We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.     

p21-Activated Kinase 2 Regulates Endothelial Development and Function through the Bmk1/Erk5 Pathway

p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.     

The Aurora/Ipl1p kinase family: regulators of chromosome segregation and cytokinesis.

Members of the Aurora/Ipl1p family of mitotically regulated serine/threonine kinases are emerging as key regulators of chromosome segregation and cytokinesis. Proper chromosome segregation and cytokinesis ensure that each daughter cell receives the full complement of genetic material. Defects in these processes can lead to aneuploidy and the propagation of genetic abnormalities. This review discusses the Aurora/Ipl1p kinases in terms of their protein structure and proposed function in mitotic cells and also the potential role of aurora2 in human cancer.     

Emerging functions of p21-activated kinases in human cancer cells

The p21 activated kinases (Paks), an evolutionarily conserved family of serine/threonine kinases, are important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, and angiogenesis. Paks are widely expressed in numerous tissues and are activated by growth factors and extracellular signals through GTPase-dependent and -independent mechanisms. Overexpression of Paks in epithelial cancer cells has been shown to increase migration potential, increase anchorage independent growth, and cause abnormalities in mitosis. Dysregulation of Paks has been reported in several human tumors and neurodegenerative diseases. A growing list of novel Pak interacting proteins has opened up exciting avenues of investigation by which to understand the functions of Paks in tumorigenesis. In this review, we will summarize the current knowledge of the Paks family with respect to emerging cellular functions and possible contributions to cancer.     

Septin1, a new interaction partner for human serine/threonine kinase aurora-B

Several families of kinases work together to ensure the rate and precision of mitosis. Aurora-B is an important serine/threonine kinase required for chromosome segregation and cytokinesis. Identification of Aurora-B substrates will help to enhance our understanding of the molecular mechanism of mitosis. Through a yeast two-hybrid screen, we found a novel partner of Aurora-B, Septin1, belonging to a conserved family of GTPase proteins that localize to the cleavage furrow and are involved in cytokinesis. We confirmed this interaction using Co-immunoprecipitation experiments in mammalian cells and GST-pull-down analysis in vitro. Moreover, Aurora-B can phosphorylate Septin1 in vitro. We identified that Ser248, Ser307, and Ser315 are the main phosphorylation sites in Septin1. These two proteins partially co-localize to the midbody during cytokinesis. So, it is possible that Septin1's role in the regulation of cytokinesis is related to its phosphorylation by Aurora-B. Unlike previous reports that Septins function in cytokinesis and localize to the cleavage furrow, we found that Septin1 localizes to the spindle pole throughout mitosis, indicating that Septin1 may function in chromosome segregation as well.     

Polo-like kinase-activating kinases: Aurora A,Aurora B and what else?

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G₂, and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.     

A p21-activated kinase-controlled metabolic switch up-regulates phagocyte NADPH oxidase

Chemoattractant-stimulated phagocytes increase their glucose uptake and divert energy production from glycolysis to the pentose phosphate pathway to generate NADPH. NADPH is a required cofactor for the NADPH oxidase to produce reactive oxygen metabolites, an important microbicidal tool in host defense. p21-Activated kinases (Paks) are regulated by the GTPases Rac and Cdc42 and control actin dynamics and phosphorylation of the oxidase component p47(phox). Here we report the interaction of Pak with phosphoglycerate mutase (PGAM)-B, an enzyme of the glycolytic pathway. Activated Pak1 inhibits glycolysis by association of its catalytic domain with PGAM-B and subsequent phosphorylation of the enzyme on serine residues 23 and 118, thereby abolishing PGAM activity. Leukocyte activation through chemoattractant receptors leads to Pak activation and transient inhibition of endogenous PGAM-B activity. Consistent with these observations, treatment of neutrophils with phosphoglycolic acid, a competitive PGAM-B inhibitor, increases upstream intermediates, thereby amplifying the respiratory burst. These results demonstrate that Rho GTPases regulate the glycolytic pathway through Pak and suggest a link between chemoattractant signaling and metabolic responses to enhance host defense.     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53^−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.Key words: Aurora A kinase, polyploidy, apoptosis, p53, cell cycle     

Identification of an autoinhibitory domain of p21-activated protein kinase 5

The p21-activated protein kinases (Paks) are serine/threonine protein kinases activated by binding to Rho family small GTPases, Rac and Cdc42. Recently, Pak family members have been subdivided into two groups, I and II. Group II Paks, including Pak4, Pak5, and Pak6, does not contain the highly conserved autoinhibitory domain that is found in the group I Paks members, i.e. Pak1, Pak2, and Pak3. In the present study, we have purified the glutathione S-transferase fusion form of Pak5 and shown for the first time that Pak5 autophosphorylation can be activated by GTP bound form of Cdc42. Mutation of histidine residues 19 and 22 to leucine on the p21-binding domain of Pak5 completely abolished the binding of Cdc42 and the Cdc42-mediated autophosphorylation. On the other hand, mutation of tyrosine 40 to cysteine of Cdc42 did not knockout the binding of Pak5. Analysis of C-terminal deletion mutants has identified an autoinhibitory fragment of Pak5 that is absent from other group II Pak family members. Taken together, these results suggest that Pak5, like Pak1, contains an autoinhibitory domain and its activity is regulated by Cdc42.     

Pak1 phosphorylation on t212 affects microtubules in cells undergoing mitosis

The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Recently, overexpression of Xenopus Pak5 was shown to enhance microtubule stabilization, and it was shown that mammalian Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. We have identified a specific phosphorylation site on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO(4)), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Here, we show that cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO(4)) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division.