Unusual phyletic distribution of peptidases as a tool for identifying potential drug targets

Profound changes in the phosphorylation state of many proteins occur during mitosis. It is well established that many of these mitotic phosphorylations are carried out by archetypal mitotic kinases that are activated only during mitosis, shifting the equilibrium of kinases and phosphatases towards phosphorylation. However, many studies have also detailed the phosphorylation of proteins at mitosis by kinases that are constitutively active throughout the cell cycle. In most cases, it is uncertain how kinases and phosphatases that appear to be constitutively active can induce phosphorylations specifically at mitosis. In this issue of the Biochemical Journal, Escargueil and Larsen provide evidence of an interesting alternative mechanism to attain specific mitotic phosphorylation. A mitosis-specific phosphorylation site in DNA topoisomerase IIalpha, which is recognized by the MPM-2 antibody, is phosphorylated by protein kinase CK2. The authors found that phosphorylation of this site is suppressed during interphase due to competing dephosphorylation by protein phosphatase 2A. Interestingly, protein phosphatase 2A is excluded from the nucleus during early mitosis, allowing CK2 to phosphorylate topoisomerase IIalpha. It is possible that similar mechanisms are used to regulate the phosphorylation of other proteins.     

Kinases regulating Golgi apparatus structure and function

Protein kinases control Golgi function in both mitotic and interphase cells. In mitosis, phosphorylation of structural proteins by Cdk1 (cyclin-dependent kinase 1)-cyclin B, Polo-like and mitogen-activated protein kinases underlie changes in Golgi reorganization during cell division. While in interphase, signalling pathways that are associated with the Golgi control secretory function through a variety of mechanisms. Some of these, notably those involving protein kinase D and Ste20 family kinases, are also relevant for the establishment and maintenance of cell polarization and migration.     

Structural features of the protein kinase domain and targeted binding by small-molecule inhibitors

Protein kinases are key components in cellular signaling pathways as they carry out the phosphorylation of proteins, primarily on Ser, Thr, and Tyr residues. The catalytic activity of protein kinases is regulated, and they can be thought of as molecular switches that are controlled through protein–protein interactions and post-translational modifications. Protein kinases exhibit diverse structural mechanisms of regulation and have been fascinating subjects for structural biologists from the first crystal structure of a protein kinase over 30 years ago, to recent insights into kinase assemblies enabled by the breakthroughs in cryo-EM. Protein kinases are high-priority targets for drug discovery in oncology and other disease settings, and kinase inhibitors have transformed the outcomes of specific groups of patients. Most kinase inhibitors are ATP competitive, deriving potency by occupying the deep hydrophobic pocket at the heart of the kinase domain. Selectivity of inhibitors depends on exploiting differences between the amino acids that line the ATP site and exploring the surrounding pockets that are present in inactive states of the kinase. More recently, allosteric pockets outside the ATP site are being targeted to achieve high selectivity and to overcome resistance to current therapeutics. Here, we review the key regulatory features of the protein kinase family, describe the different types of kinase inhibitors, and highlight examples where the understanding of kinase regulatory mechanisms has gone hand in hand with the development of inhibitors.     

Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases

Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal alphaC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases.     

古菌蛋白激酶的研究进展

磷酸化是蛋白质翻译后修饰(post-translational modification)的主要方式,可由蛋白激酶、磷酸转移酶、磷酸化酶等多种方式催化进行。其中,由蛋白激酶(protein kinases)/磷酸酶(protein phosphatases)介导的可逆的蛋白磷酸化是细胞中信号转导的重要机制,在DNA复制、转录、蛋白质翻译、DNA损伤修复等生命过程中起广泛的调节作用。目前,古菌中蛋白激酶的研究尚属于初期阶段。虽然磷酸化蛋白质组学研究表明,古菌中存在大量的磷酸化蛋白质,但是我们对其具体催化作用的酶及调控机制尚不清楚。本文总结了古菌中已报道的蛋白激酶所参与的生命过程,包括古菌的DNA代谢、细胞代谢、细胞周期和运动机制等四个方面,并对今后的研究提出展望。     

Phosphoproteome dynamics during mitotic exit in budding yeast

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.     

Activation of calcium/calmodulin regulated kinases.

Among numerous protein kinases found in mammalian cell systems there is a distinct subfamily of serine/threonine kinases that are regulated by calmodulin or other related activators in a calcium concentration dependent manner. Members of this family are involved in various cellular processes like cell proliferation and death, cell motility and metabolic pathways. In this contribution we shall review the available structural biology data on five members of this kinase family (calcium/calmodulin dependent kinase, twitchin kinase, titin kinase, phosphorylase kinase, myosin light chain kinase). As a common element, all these kinases contain a regulatory tail, which is C-terminal to their catalytic domain. The available 3D structures of two members, the serine/threonine kinases of the giant muscle proteins twitchin and titin in the autoinhibited conformation, show how this regulatory tail blocks their active sites. The structures suggest that activation of these kinases requires unblocking the active site from the C-terminal extension and conformational rearrangement of the active site loops. Small angle scattering data for myosin light chain kinase indicate a complete release of the C-terminal extension upon calcium/calmodulin binding. In addition, members of this family are regulated by diverse add-on mechanisms, including phosphorylation of residues within the activation segment or the P+1 loop as well as by additional regulatory subunits. The available structural data lead to the hypothesis of two different activation mechanisms upon binding to calcium sensitive proteins. In one model, the regulatory tail is entirely released ("fall-apart"). The alternative model ("looping-out") proposes a two-anchored release mechanism.     

Casein kinase-2-type protein kinases in plants: possible targets of polyamine action during growth regulation?

The biochemical mechanisms by which polyamines influence plant growth and development are not known. One of mechanisms frequently proposed is that polyamines can bind to key cellular enzymes and modulate their activity. Polyamines have been reported to alter the activity of a number of enzymes in vitro. Among these the casein kinase-2 protein kinases are of particular interest, not only because of increasing recognition of the major role of protein phosphorylation in regulating plant cell metabolism, but also because these kinases have been specifically implicated in the phosphorylation of trans-acting factors and thus could regulate gene expression. Casein kinase-2-type protein kinases have been purified and characterized from both plants and animals. Their structural and biochemical properties appear to have been remarkably conserved throughout evolution. Most are stimulated by mM levels of polyamines. Although this concentration is within the range estimated to occur in plant cells, not enough is known about [polyamine] in subcellular compartments and about how rapidly this concentration can be altered by hormonal and environmental signals to predict whether polyamines play a major role in the regulation of casein kinase-2 protein kinase activity in vivo.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4100 and > 2200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.     

How protein kinases co-ordinate mitosis in animal cells

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.     

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with, a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.     

Protein Kinases in Neutrophils: A Review

In chemotactic factor-stimulated neutrophils, rapid increases of intracellular levels of cyclic AMP, calcium, and diacylglycerol have been observed and may be linked to protein kinase activation. The study of the physiological role and regulation of protein kinases in the neutrophil and the identification of their substrates has provided valuable information on the molecular mechanism of neutrophil activation. The focus of this review is on those aspects of protein kinases that are relevant to neutrophil activation and on the substrate proteins for these protein kinases. The possible role of protein phosphorylation in neutrophil function is also discussed.     

Structure and function of the BAH domain in chromatin biology

Abstract

Proteins containing Bromo Adjacent Homology (BAH) domain are often associated with biological processes involving chromatin, and mutations in BAH domains have been found in human diseases. A number of structural and functional studies have revealed that the BAH domain plays diverse and versatile roles in chromatin biology, including protein–protein interactions, recognition of methylated histones and nucleosome binding. Here we review recent developments in structural studies of the BAH domain, and intend to place the structural results in the context of biological functions of the BAH domain-containing proteins. A converging theme from the structural studies appears that the predominantly β-sheet fold of the BAH domain serves as a scaffold, and function-specific structural features are incorporated at the loops connecting the β-strands and surface-exposed areas. The structures clearly specified regions critical for protein–protein interactions, located the position of methyllysine-binding site and implicated areas important for nucleosome binding. The structural results provided valuable insights into the molecular mechanisms of BAH domains in molecular recognitions, and the information should greatly facilitate mechanistic understanding of BAH domain proteins in chromatin biology.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x_L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b₆f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studies is that of a complex regulatory network of chloroplast protein kinases and phosphatases that is involved in light acclimation, in maintenance of the plastoquinone redox poise under fluctuating light and in the adjustment to metabolic needs.     

Protein kinases in control of the centrosome cycle.

The centrosome is the major microtubule nucleating center of the animal cell and forms the two poles of the mitotic spindle upon which chromosomes are segregated. During the cell division cycle, the centrosome undergoes a series of major structural and functional transitions that are essential for both interphase centrosome function and mitotic spindle formation. The localization of an increasing number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling many of these transitions. Here, we focus on two protein kinases, the polo-like kinase 1 and the NIMA-related kinase 2, for which recent data indicate key roles during the centrosome cycle.