VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.     

Regulation of Plasmodium falciparum Pfnek3 relies on phosphorylation at its activation loop and at threonine 82

A mitogen-activated protein kinase (MAPK), Pfmap2, has been identified in Plasmodium falciparum. However, its bona fide activator remains elusive as no MAPK kinase (MAPKK) homologues have been found so far. Instead, Pfnek3, a NIMA (never in mitosis, Aspergillus)-related kinase, was earlier reported to display a MAPKK-like activity due to its activating effect on Pfmap2. In this study, the regulatory mechanism of Pfnek3 was investigated. Pfnek3 was found to possess a SSEQSS motif within its activation loop that fulfills the consensus SXXXS/T phospho-activating sequence of MAPKKs. Functional analyses of the SSEQSS motif by site-directed mutagenesis revealed that phosphorylation of residues S221 and S226 is essential for mediating Pfnek3 activity. Moreover, via tandem mass-spectrometry, residue T82 was uncovered as an additional phosphorylation site involved in Pfnek3 activation. Collectively, these results provide valuable insights into the potential in vivo regulation of Pfnek3, with residues T82, S221 and S226 functioning as phospho-activating sites.     

Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family. Received 17 July 2007; received after revision 14 August 2007; accepted 12 September 2007     

Polyamine-dependent gene expression

The polyamines spermidine and spermine along with the diamine putrescine are involved in many cellular processes, including chromatin condensation, maintenance of DNA structure, RNA processing, translation and protein activation. The polyamines influence the formation of compacted chromatin and have a well-established role in DNA aggregation. Polyamines are used in the posttranslational modification of eukaryotic initiation factor 5A, which regulates the transport and processing of specific RNA. The polyamines also participate in a novel RNA-decoding mechanism, a translational frameshift, of at least two known genes, the TY1 transposon and mammalian antizyme. Polyamines are crucial for their own regulation and are involved in feedback mechanisms affecting both polyamine synthesis and catabolism. Recently, it has become apparent that the polyamines are able to influence the action of the protein kinase casein kinase 2. Here we address several roles of polyamines in gene expression.Received 27 November 2002; received after revision 9 January 2003; accepted 31 January 2003     

Ubiquitin-proteasome system

The yeast Saccharomyces cerevisiae has turned out to be an invaluable tool in the molecular biological sciences for elucidating the housekeeping functions of eukaryotic cells. Due to its easy amenability to biochemical, genetic, molecular biological and cell biological experimentation, including genomics and proteomics, yeast has become one of the most frequently used eukaryotic model organisms. One of the fields where studies in yeast have a truly pacemaking character is cellular control by proteolysis. The function of vacuolar (lysosomal) proteolysis was elucidated. The in vivo role of ubiquitin and its relation to the proteasome was uncovered. This research led to an avalanche of studies in many different eukaryotic systems, including mammals, and provided us with surprising new insights in cellular control in health and disease.     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Genetic analysis of ubiquitin-dependent protein degradation.

Selective degradation of cellular proteins serves to eliminate abnormal proteins and to mediate the turnover of certain short-lived proteins, many of which have regulatory functions. In eukaryotes a major pathway for selective protein degradation is ATP-dependent and is mediated by the ubiquitin system. This pathway involves substrate recognition by components of a ubiquitin-protein ligase system, covalent attachment of ubiquitin moieties to proteolytic substrates, and subsequent degradation of these conjugates by a multicatalytic protease complex. Recent genetic evidence suggests that the remarkable selectivity of this process is largely controlled at the level of substrate recognition by the ubiquitin ligase system. In Saccharomyces cerevisiae, ubiquitin-conjugating enzymes UBC1, UBC4 and UBC5 have been identified as key components of this highly conserved degradation pathway. Genetic analysis indicates that ubiquitin-dependent proteolysis is essential for cell viability and that UBC4 and UBC5 enzymes are essential components of the eukaryotic stress response.     

The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum

Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum, several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium. Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.     

TPX2: of spindle assembly,DNA damage response,and cancer

For more than 15 years, TPX2 has been studied as a factor critical for mitosis and spindle assembly. These functions of TPX2 are attributed to its Ran-regulated microtubule-associated protein properties and to its control of the Aurora A kinase. Overexpressed in cancers, TPX2 is being established as marker for the diagnosis and prognosis of malignancies. During interphase, TPX2 resides preferentially in the nucleus where its function had remained elusive until recently. The latest finding that TPX2 plays a role in amplification of the DNA damage response, combined with the characterization of TPX2 knockout mice, open new perspectives to understand the biology of this protein. This review provides an historic overview of the discovery of TPX2 and summarizes its cytoskeletal and signaling roles with relevance to cancer therapies. Finally, the review aims to reconcile discrepancies between the experimental and pathological effects of TPX2 overexpression and advances new roles for compartmentalized TPX2.     

Novel actions of tyrphostin AG 879: inhibition of RAF-1 and HER-2 expression combined with strong antitumoral effects on breast cancer cells

Binding of growth factors to cell surface receptors activates protein tyrosine kinases (PTKs) that initiate cascades of downstream signaling events including the mitogen-activated protein (MAP) kinase cascade. This study reports that the PTK inhibitor AG 879 inhibits proliferation of human breast cancer cells through an effect involving inhibition of MAP kinase activation, but which cannot be explained by effects of AG 879 on its known PTK targets. Instead, AG 879 markedly inhibits expression of the RAF-1 gene, which encodes an upstream MAP kinase kinase kinase. Additionally, expression of HER-2, but not of other genes tested, is inhibited by this compound. These novel effects have to be considered when using AG 879 as a TRK-A and HER-2 inhibitor but may have useful therapeutic implications.     

Adducin: structure, function and regulation

Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either alpha/beta or alpha/gamma heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.     

Functions of actin in endocytosis

Endocytosis is a fundamental eukaryotic process required for remodelling plasma-membrane lipids and protein to ensure appropriate membrane composition. Increasing evidence from a number of cell types reveals that actin plays an active, and often essential, role at key endocytic stages. Much of our current mechanistic understanding of the endocytic process has come from studies in budding yeast and has been facilitated by yeast’s genetic amenability and by technological advances in live cell imaging. While endocytosis in metazoans is likely to be subject to a greater array of regulatory signals, recent reports indicate that spatiotemporal aspects of vesicle formation requiring actin are likely to be conserved across eukaryotic evolution. In this review we focus on the ‘modular’ model of endocytosis in yeast before highlighting comparisons with other cell types. Our discussion is limited to endocytosis involving clathrin as other types of endocytosis have not been demonstrated in yeast.     

Functions of reticulons in plants: What we can learn from animals and yeasts

Reticulons (RTNs) are membrane-spanning proteins sharing a typical domain named reticulon homology domain (RHD). RTN genes have been identified in all eukaryotic organisms examined so far, and the corresponding proteins have been found predominantly associated to the endoplasmic reticulum membranes. In animal and yeast, in which knowledge of the protein family is more advanced, RTNs are involved in numerous cellular processes such as apoptosis, cell division and intracellular trafficking. Up to now, a little attention has been paid to their plant counterparts, i.e., RTNLBs. In this review, we summarize the data available for RTNLB proteins and, using the data obtained with animal and yeast models, several functions for RTNLBs in plant cells are proposed and discussed. Received 01 July 2008; received after revision 08 September 2008; accepted 30 September 2008     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.     

Autophagy: molecular mechanisms,physiological functions and relevance in human pathology

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Over the last years, yeast genetic screens have considerably increased our knowledge about the molecular mechanisms of autophagy, and a number of genes involved in fundamental steps of the autophagic pathway have been identified. Most of these autophagy genes are present in higher eukaryotes indicating that this process has been evolutionarily conserved. In yeast, autophagy is mainly involved in adaptation to starvation, but in multicellular organisms this route has emerged as a multifunctional pathway involved in a variety of additional processes such as programmed cell death, removal of damaged organelles and development of different tissue-specific functions. Furthermore, autophagy is associated with a growing number of pathological conditions, including cancer, myopathies and neurodegenerative disorders. The physiological and pathological roles of autophagy, as well as the molecular mechanisms underlying this multifunctional pathway, are discussed in this review.Received 12 January 2004; received after revision 29 January 2004; accepted 4 February 2004     

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.