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BBA - Proteins and Proteomics (v.1754, #1-2)

Editorial Board (pp. ii).

Special issue—Inhibitors of protein kinases by David Shugar (pp. 1-2).

Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia by Paul William Manley; Sandra W. Cowan-Jacob; J�rgen Mestan (pp. 3-13).

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.

Keywords: Bcr-Abl; Chronic myeloid leukaemia; Protein kinase inhibitor; Crystal structure; AMN107; BMS-354825; AZD0530

The RIO kinases: An atypical protein kinase family required for ribosome biogenesis and cell cycle progression by Nicole LaRonde-LeBlanc; Alexander Wlodawer (pp. 14-24).

Atypical protein kinases (aPKs) include proteins known to be involved in the phosphorylation-mediated regulation of a wide variety of cellular processes, as well as some for which the function is, as yet, unknown. At present, 13 families of aPKs have been identified in the human genome. This review briefly summarizes their known properties, but concentrates in particular on the RIO family of aPKs. Representatives of this family are present in organisms varying from archaea to humans. All these organisms contain at least two RIO proteins, Rio1 and Rio2, but a third Rio3 group is present in multicellular eukaryotes. Crystal structures of A. fulgidus Rio1 and Rio2 have shown that whereas the overall fold of these enzymes resembles typical protein kinases, some of the kinase structural domains, particularly those involved in peptide substrate binding, are not present. The mode of binding of nucleotides also differs from other kinases. While the enzymatic activity of Rio1 and Rio2 has been demonstrated and both have been shown to be essential in S. cerevisiae and required for proper cell cycle progression and chromosome maintenance, the biological substrates of RIO proteins still remain to be identified.

Keywords: Atypical protein kinase; Structure; ATP binding; Enzymatic activity; Ribosome biogenesis; Rio1; Rio2

Dynamics of signaling by PKA by Susan S. Taylor; Choel Kim; Dominico Vigil; Nina M. Haste; Jie Yang; Jian Wu; Ganesh S. Anand (pp. 25-37).

The catalytic and regulatory subunits of cAMP-dependent protein kinase (PKA) are highly dynamic signaling proteins. In its dissociated state the catalytic subunit opens and closes as it moves through its catalytic cycle. In this subunit, the core that is shared by all members of the protein kinase family is flanked by N- and C-terminal segments. Each are anchored firmly to the core by well-defined motifs and serve to stabilize the core. Protein kinases are not only catalysts, they are also scaffolds. One of their major functions is to bind to other proteins. In addition to its interactions with the N- and C- termini, the catalytic subunit interacts with its inhibitor proteins, PKI and the regulatory subunits. Both bind with subnanomolar affinity. To achieve this tight binding requires docking of a substrate mimetic to the active site cleft as well as a peripheral docking site. The peripheral site used by PKI is distinct from that used by RIα as revealed by a recent structure of a C:RIα complex. Upon binding to the catalytic subunit, the linker region of RIα becomes ordered. In addition, cAMP-binding domain A undergoes major conformational changes. RIα is a highly malleable protein. Using small angle X-ray scattering, the overall shape of the regulatory subunits and corresponding holoenzymes have been elucidated. These studies reveal striking and surprising isoform differences.

Keywords: Abbreviations; cAMP; cyclic-3′, 5′-adenosine monophosphate; PKA; cAMP-dependent protein kinase; PKI; heat stable protein kinase A inhibitor; PDK1; phosphoinositide-dependent kinase-1; ATP; adenosine triphosphate; ADP; adenosine diphosphate; D/D domain; dimerization/docking domain; SAXS; small-angle X-ray scattering; AKIP1; A Kinase Interacting Protein 1; H/DMS; Hydrogen/deuterium exchange coupled with mass spectrometry; PBC; phosphate binding cassettePKA; PKI; Holoenzyme; RIα; RIIα; RIIβ; Signaling; Allostery; Dynamics

Crystallography for protein kinase drug design: PKA and SRC case studies by Christine B. Breitenlechner; Dirk Bossemeyer; Richard A. Engh (pp. 38-49).

Protein crystallography can be used throughout the drug discovery process to obtain diverse information critical for structure based drug design. At a minimum, a single target structure may be available. Optimally, and especially for protein kinases, a broad range of crystal structures should be obtained to characterize target flexibility, structure modulation via co-factor binding or posttranslational modification, ligand induced conformational changes, and off-target complex structures for selectivity optimization. The flexibility of the protein kinases is in contrast to the need for “crystallizable�? constructs, that is, proteins that crystallize under varying conditions and in varying crystal packing arrangements. Strategies to produce crystallizable protein kinase constructs include truncation to the catalytic domain, co-crystallization with rigidifying ligands, crystallization of known rigid forms, and point mutation to improve homogeneity or mimic less crystallizable proteins. PKA, the prototypical serine/threonine protein kinase, and SRC, a tyrosine kinase and the first identified oncoprotein, provide multiple examples of these various approaches to protein kinase crystallography for drug design.

Keywords: Abbreviations; PKA; Protein kinase A; PIFtide; Phosphoinositide-dependent kinase 1 interacting fragment peptide; CDK2; cyclin-dependent kinase 2; ATP; adenosine triphosphate; PKB; Protein kinase B; PKC; Protein Kinase C; PKG; Protein kinase G; PKI; protein kinase A inhibitor; LCK; Lymphocyte specific protein tyrosine kinase; PKAB4; four-fold PKB-like mutant of PKASRC; PKA; Crystal structure; Drug design; Protein kinase inhibitor; Flexibility

Signaling protein inhibitors via the combinatorial modification of peptide scaffolds by David S. Lawrence (pp. 50-57).

Compounds that selectively interfere with protein–protein interactions are not only invaluable as biological reagents, but may ultimately serve as therapeutically useful drugs for the treatment of a wide variety of disease states. However, unlike active site directed inhibitors that bind to a relatively small, well-defined, hydrophobic pocket, reagents that disrupt protein–protein interactions must contend with a protein surface that is comparatively large, ill defined, and solvent exposed. We have developed a straightforward method for the acquisition of protein–protein interaction inhibitors. The library-based strategy starts with low affinity consensus sequence peptides, which are then transformed in a stepwise fashion into high affinity inhibitors. The approach has been used to create potent ligands for SH2 and SH3 domains, as well as powerful and highly selective inhibitors for protein kinases and phosphatases. The protocol is easily automated and therefore has the potential to be routinely applied, in a high throughput fashion.

Keywords: Signal transduction; Inhibitor; Peptide; Combinatorial library; High throughput screening; Protein–Protein interaction

Exploiting structural principles to design cyclin-dependent kinase inhibitors by Martin Noble; Paul Barrett; Jane Endicott; Louise Johnson; Jim McDonnell; Giles Robertson; Alex Zawaira (pp. 58-64).

Although cyclin-dependent kinases (CDKs) have been extensively targeted in anti cancer drug design, no CDK inhibitor has yet been approved for use in cancer therapy. While this may in part be because inhibitors clinically evaluated to date have not demonstrated clean inhibition of a single CDK, another contributing factor is an apparent latent functional redundancy in the CDK cell-cycle regulatory system. This further complicates the already challenging goal of targeting CDKs, since it implies that a therapeutically useful inhibitor will have to selectively inhibit more than one CDK family member among the complement of cellular proteins. Despite these difficulties, achieving an appropriate profile of CDK inhibition may yet be possible using ATP-competitive inhibitors, thanks to advances in computational and experimental methods of drug design. However, as an alternative to ATP-competitive inhibitors, inhibitors that interfere with a CDK-specific protein:protein interaction, such as that which occurs at the recruitment site found on several cyclins, may offer a route to a therapeutically useful inhibitory profile.

Keywords: Abbreviations; CDK; cyclin-dependent kinase; PKA; cAMP-dependent protein kinase; CMGC-I; CDK-like protein kinases; CML; Chronic myelogenous leukaemia; PfPK5; Plasmodium falciparum; Protein Kinase 5Cyclin-dependent kinase inhibitor; CDK; Drug design; Protein dynamic; Protein:protein interaction; Peptide inhibitor

Protein kinase structure and function analysis with chemical tools by Kui Shen; Aliya C. Hines; Dirk Schwarzer; Kerry A. Pickin; Philip A. Cole (pp. 65-78).

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase–substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide–ATP conjugates into protein structures. Using expressed protein ligation, a Src–ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2–phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.

Keywords: Abbreviations; AANAT; serotonin N-acetyltransferase or arylalkylamine N-acetyltransferase; aPP; avian pancreatic polypeptide; CaMKII; Ca; ²⁺; /calmodulin kinase II; CAT; chloramphenicol acetyltransferase; CHO; Chinese hamster ovary; Csk; C-terminal Src kinase; Fmoc; fluorenylmethoxycarbonyl; F; ₂; Pmp; phosphonodifluoromethylene phenylalanine; Grb2; growth factor receptor-bound protein 2; GST; glutathione S-transferase; IRK; insulin receptor kinase; MALDI; matrix-assisted laser desorption/ionization; MESNA; 2-mercaptoethanesulfonic acid; Pfa; phosphonodifluoromethylene alanine; PKB; protein kinase B or Akt kinase; PKG; cGMP-dependent protein kinase; PKI; protein kinase inhibitor protein; Pma; phosphonomethylene alanine; Pmp; phosphonomethylene phenylalanine; pSer; phosphoserine; pThr; phosphothreonine; PTK; protein tyrosine kinase; PTP; protein tyrosine phosphatase; pTyr; phosphotyrosine; SH2; Src homology 2; SHP; SH2(-containing) phosphatase; SRE; serum response element; tBu; tert-butyl; TCEP; tris(2-carboxyethyl)phosphine; TOF; time of flightBisubstrate kinase inhibitor; Expressed protein ligation; Protein semisynthesis; Nonhydrolyzable phosphonate analog; Phosphotyrosine; Phosphoserine

Peptide inhibitors of protein kinases—discovery, characterisation and use by Marie A. Bogoyevitch; Renae K. Barr; Albert J. Ketterman (pp. 79-99).

Protein kinases are now the second largest group of drug targets, and most protein kinase inhibitors in clinical development are directed towards the ATP-binding site. However, these inhibitors must compete with high intracellular ATP concentrations and they must discriminate between the ATP-binding sites of all protein kinases as well the other proteins that also utilise ATP. It would therefore be beneficial to target sites on protein kinases other than the ATP-binding site. This review describes the discovery, characterisation and use of peptide inhibitors of protein kinases. In many cases, the development of these peptides has resulted from an understanding of the specific protein-binding partners for a particular protein kinase. In addition, novel peptide sequences have been discovered in library screening approaches and have provided new leads in the discovery and/or design of peptide inhibitors of protein kinases. These approaches are therefore providing exciting new opportunities in the development of ATP non-competitive inhibitors of protein kinases.

Keywords: Abbreviations; AKAP; A-Kinase Anchoring Protein; EGF-R; Epidermal Growth Factor-Receptor; GSK; Glycogen Synthase Kinase; IC; ₅₀; concentration of compound to achieve 50% inhibition; JIP; JNK Interacting Protein; JNK; c-Jun N-terminal Kinase; K; _i; inhibition constant; MLCK; Myosin Light Chain Kinase; PKC; Protein Kinase C; PKI; Protein Kinase A Inhibitor; RACK; Receptor for Activated C-Kinase; SOCS; Suppressor of Cytokine Signalling; TI-JIP; truncated inhibitory region of JIPPeptide inhibitor; Endogenous inhibitor; Pseudosubstrate; Library screening; Peptide design; Small molecule ATP non-competitive inhibitor

Functional studies of protein tyrosine phosphatases with chemical approaches by Zhong-Yin Zhang (pp. 100-107).

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTP activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases. A number of PTPs have been identified as novel therapeutic targets for the treatment of various diseases. However, because PTPs can both enhance and antagonize PTK signaling, it is essential to elucidate the physiological context in which PTPs function. Assigning the functional significance of PTPs in normal physiology and in diseases remains a major challenge in cell signaling. Efficient methodologies are needed to delineate the PTP functions. One strategy is to apply chemical genetic approaches utilizing potent and selective PTP inhibitors to study the physiological roles of the PTPs in vivo. Recent work using this approach to define the roles of PTP1B in insulin- and integrin-mediated processes is discussed. Another strategy is to apply activity-based proteomic techniques to measure globally PTP activity in both normal and pathological conditions. The ability to profile the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets. Recent development on the design and characterization of activity-based PTP probes is highlighted.

Keywords: Abbreviations; PTP; protein tyrosine phosphatase; PTK; protein tyrosine kinase; FAK; focal adhesion kinase; ERK; extracellular signal-regulated kinase; p130; ^Cas; Crk associated substrate.Protein tyrosine phosphatase; PTP1B; Protein tyrosine phosphatase inhibitor; Chemical genetic; Protein tyrosine phosphatase probe; Activity-based proteomic

Involvement of the small protein tyrosine phosphatases TC-PTP and PTP1B in signal transduction and diseases: From diabetes, obesity to cell cycle, and cancer by Nadia Dub�; Michel L. Tremblay (pp. 108-117).

As in other fields of biomedical research, the use of gene-targeted mice by homologous recombination in embryonic stem cells has provided important findings on the function of several members of the protein tyrosine phosphatase (PTP) family. For instance, the phenotypic characterization of knockout mice has been critical in understanding the sites of action of the related PTPs protein tyrosine phosphatase 1B (PTP1B) and T-cell-PTP (TC-PTP). By their increased insulin sensitivity and insulin receptor hyperphosphorylation, PTP1B null mice demonstrated a clear function for this enzyme as a negative regulator of insulin signaling. As well, TC-PTP has also been recently involved in insulin signaling in vitro. Importantly, the high identity in their amino acid sequences suggests that they must be examined simultaneously as targets of drug development. Indeed, they possess different as well as overlapping substrates, which suggest complementary and overlapping roles of both TC-PTP and PTP1B. Here, we review the function of PTP1B and TC-PTP in diabetes, obesity, and processes related to cancer.

Keywords: Abbreviations; EGFR; epidermal growth factor receptor; GHR; growth hormone receptor; IGF-IR; insulin-like growth factor I receptor; IR; insulin receptor; IRS; insulin receptor substrate; JAK; janus kinase; KO; knockout; LeptinR; leptin receptor; PDGFR; platelet derived growth factor receptor; PKB; protein kinase B; PTKs; protein tyrosine kinases; PTPs; protein tyrosine phosphatases; PTP1B; protein tyrosine phosphatase 1B; TC-PTP; T-cell protein tyrosine phosphatase; STAT; signal transducers and activators of transcription; WT; wild-typeTyrosine phosphatase; Signaling; Diabetes; Obesity; Cancer

P-Ser-HPr—a link between carbon metabolism and the virulence of some pathogenic bacteria by Josef Deutscher; Rana Herro; Alexa Bourand; Ivan Mijakovic; Sandrine Poncet (pp. 118-125).

HPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of PrfA. In an hprKV267F mutant, in which most of the HPr is transformed into P-Ser-HPr, PrfA was barely active. The ptsH1 mutation (Ser-46 of HPr replaced with an alanine) prevented the inhibitory effect of the hprKV267F mutation. Interestingly, disruption of ccpA also inhibited PrfA activity. This effect is probably also mediated via P-Ser-HPr, since ccpA disruption leads to elevated amounts of P-Ser-HPr. Indeed, a ccpA ptsH1 double mutant exhibited normal PrfA activity. In S. pyogenes, the expression of several virulence genes depends on the transcription activator Mga. Interestingly, the mga promoter is preceded by an operator site, which serves as target for the CcpA/P-Ser-HPr complex. Numerous Gram-negative pathogens also contain hprK, which is often organised in an operon with transcription regulators necessary for the development of virulence, indicating that in these organisms P-Ser-HPr also plays a role in pathogenesis. Indeed, inactivation of Neisseria meningitidis hprK strongly diminished cell adhesion of this pathogen.

Keywords: Abbreviations; CCR; carbon catabolite repression; CcpA; catabolite control protein A; Crh; catabolite repression HPr; FHA domain; forkhead-associated domain; FBP; fructose-1,6-bisphosphate; HprK/P; HPr kinase/phosphorylase; PEP; phosphoenolpyruvate; PTS; phosphoenolpyruvate:carbohydrate phosphotransferase system; P-Ser-HPr; seryl-phosphorylated HPr; P∼His-HPr; histidyl-phosphorylated HPr Listeria monocytogenes; Catabolite repression; PrfA; HPr kinase/phosphorylase; Mga; Protein phosphorylation

The bacterial HPr kinase/phosphorylase: A new type of Ser/Thr kinase as antimicrobial target by Sylvie Nessler (pp. 126-131).

Protein phosphorylation plays a major role in bacterial cellular regulation as in eukaryotes. The HPr Kinase/Phosphorylase (HprK/P) was the first bacterial serine protein kinase to have had its structure determined, establishing that it is unrelated to the eukaryotic kinases. HprK/P belongs to another large structural family, the P-loop containing proteins. Among them, P-loop containing kinases have been assumed to only phosphorylate small molecules, but the example of HprK/P suggests that some may have proteins as substrates, defining novel cellular signal transduction pathways. Another major result of the studies presented here is that HprK/P also catalyses the phosphorolysis of the phosphoserine, yielding serine and pyrophosphate. The two different catalytic activities are carried out at the same active site. The determination of the structure of the complex with the protein substrates HPr and PserHPr allowed us to propose a catalytic mechanism. Since regulation of HPr phosphorylation has been shown to be involved in the virulence process of pathogenic bacteria, a search for specific inhibitors of HprK/P is of clinical interest and the first hit has already been found.

Keywords: Abbreviations; HPr; Histidine phosphocarrier protein; PserHPr; seryl-phosphorylated HPr; HprK/P; HPr Kinase/Phosphorylase; P-loop; phosphate binding loop; CcpA; catabolite control protein A; Pi; inorganic phosphate; PEP; phosphoenolpyruvateHPr kinase; Crystal; Structure; Complex; Active site; Mechanism

Protein kinases as targets for antimalarial intervention: Kinomics, structure-based design, transmission-blockade, and targeting host cell enzymes by Christian Doerig; Oliver Billker; David Pratt; Jane Endicott (pp. 132-150).

The surge of interest in protein kinases as targets for chemotherapeutic intervention in a number of diseases such as cancer and neurodegenerative disorders has stimulated research aimed at determining whether enzymes of this class might also be considered as targets in the context of diseases caused by parasitic protists. Here, we present an overview of recent developments in this field, concentrating (i) on the benefits gained from the availability of genomic databases for a number of parasitic protozoa, (ii) on the emerging field of structure-aided design of inhibitors targeting protein kinases of parasitic protists, (iii) on the concept known as transmission-blockade, whereby kinases implicated in the development of the parasite in their arthropod vector might be targeted to interfere with disease transmission, and (iv) on the possibility of controlling parasitic diseases through the inhibition of host cell protein kinases that are required for the establishment of infection by the parasites.

Keywords: Abbreviations; CamK; calmodulin kinase; CDK; cyclin-dependent kinase; CDPK; calcium-dependent protein kinase; CK1; casein kinase 1; CQ; chloroquine; EPK; eukaryotic protein kinase domain; ERK; extracellular signal-regulated kinase; GSK3; glycogen synthase kinase 3; Hb; haemoglobin; HMM; hidden markov model; JNK; Jun-N-terminal kinase; KO; knock-out; MAPK; mitogen activated protein kinase; MAPKK; mitogen activated protein kinase kinase; MEK; MAPK/ERK kinase; NIMA; never in mitosis/Aspergillus; PKA; cAMP-dependent protein kinase; PKG; cGMP-dependent protein kinase; SP; sulfadoxine-pyrimethamineParasite; Protein kinase; Development; Drug discovery; Inhibitor; Plasmodium; Malaria; Trypanosoma; Toxoplasma; Theileria; Entamoeba

Protein kinases as drug targets in trypanosomes and Leishmania by Christina Naula; Marilyn Parsons; Jeremy C. Mottram (pp. 151-159).

Protein kinases represent promising drug targets for a number of human and animal diseases. The recent completion of the sequenced genomes of three human-infective trypanosomatid protozoa, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi, has allowed the kinome for each parasite to be defined as 179, 156 and 171 eukaryotic protein kinases respectively, that is about one third of the human complement. The analysis revealed that the trypanosomatids lack members of the receptor-linked or cytosolic tyrosine kinase families, but have an abundance of STE and CMGC family protein kinases likely to be involved in regulating cell cycle control, differentiation and response to stress during their complex life-cycles. In this review, we examine the prospects for exploiting differences between parasite and mammalian protein kinases to develop novel anti-parasitic chemotherapeutic agents.

Keywords: Abbreviations; CAK; cdc2-activating kinase; CDK; cyclin-dependent kinase; CKI; casein kinase I; CRK; cdc2-related kinase gene; CRK; cdc2-related kinase protein; ePK; eukaryotic protein kinase; HAT; human African trypanosomiasisProtein kinase; Cell cycle; Chemotherapy; Trypanosoma; Leishmania

Targeting malaria with specific CDK inhibitors by Jeanne A. Geyer; Sean T. Prigge; Norman C. Waters (pp. 160-170).

Cyclin-dependent protein kinases (CDKs) are attractive targets for drug discovery and efforts have led to the identification of novel CDK selective inhibitors in the development of treatments for cancers, neurological disorders, and infectious diseases. More recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. CDKs are valid targets as they function as essential regulators of cell growth and differentiation. To date, several CDKs have been characterized from the genome of the malaria-causing protozoan Plasmodium falciparum. Our approach employs experimental and virtual screening methodologies to identify and refine chemical inhibitors of the parasite CDK Pfmrk, a sequence homologue of human CDK7. Chemotypes of Pfmrk inhibitors include the purines, quinolinones, oxindoles, and chalcones, which have sub-micromolar IC₅₀ values against the parasite enzyme, but not the human CDKs. Additionally, we have developed and validated a pharmacophore, based on Pfmrk inhibitors, which contains two hydrogen bond acceptor functions and two hydrophobic sites, including one aromatic ring hydrophobic site. This pharmacophore has been exploited to identify additional compounds that demonstrate significant inhibitory activity against Pfmrk. A molecular model of Pfmrk designed using the crystal structure of human CDK7 highlights key amino acid substitutions in the ATP binding pocket. Molecular modeling and docking of the active site pocket with selective inhibitors has identified possible receptor–ligand interactions that may be responsible for inhibitor specificity. Overall, the unique biochemical characteristics associated with this protein, to include distinctive active site amino acid residues and variable inhibitor profiles, distinguishes the Pfmrk drug screen as a paradigm for CDK inhibitor analysis in the parasite.

Keywords: Abbreviations; CDKs; cyclin-dependent protein kinases; CAK; CDK activating kinase; ARMD; accelerated resistance to multiple drugs; CKI; cyclin-dependent kinase inhibitors; MAPK; mitogen activated protein kinase; P. falciparum; CDK-related kinase; Pfcrk; RNAP CTD; RNA Polymerase II carboxyl-terminal domainCyclin dependent protein kinase; Pfmrk; Plasmodium falciparum; Malaria; CDK inhibitor

Baker's yeast as a tool for the development of antifungal kinase inhibitors—targeting protein kinase C and the cell integrity pathway by J�rgen J. Heinisch (pp. 171-182).

Today, the yeast Saccharomyces cerevisiae is probably the best-studied eukaryotic organism. This review first focusses on the signaling process which is mediated by the unique yeast protein kinase C (Pkc1p) and a downstream mitogen-activated protein kinase (MAPK) cascade. This pathway ensures cellular integrity by sensing cell surface stress and controlling cell wall biosynthesis and progression through the cell cycle. The domain structure of Pkc1p is conserved from yeast to humans. A yeast system for heterologous expression of specific domains in a chimeric yeast/mammalian PKC enzyme (“domain shuffling�?) is depicted. It is also proposed how this system could be employed for the study of protein kinase inhibitors in high-throughput screens. Moreover, a reporter assay that allows a quantitative readout of the activity of the cell integrity signaling pathway is introduced. Since a variety of protein kinases take part in the signal transduction, this broadens the range of targets for potential inhibitors.

Keywords: Abbreviations; DAG; diacyl glycerol; FACS; fluorescent activated cell sorting; GAP; GTPase activating protein; GEF; GDP/GTP exchange factor; GFP; green fluorescent protein; HR1; homology region 1; MAPK(KK); mitogen activated protein kinase (kinase kinase); PKC; protein kinase C; Pkc1p; yeast protein kinase C; RTK; receptor tyrosine kinase; X-Gal; 5-bromo-4-chloro-3-indoxyl-β-; d; -galactosideS; accharomyces cerevisiae; Fungi; Signal transduction; MAP kinase; Domain shuffling; Reporter system

Characterisation of kinase-selective inhibitors by chemical proteomics by Henrik Daub (pp. 183-190).

Low-molecular-weight inhibitors of protein kinases are extensively used as research tools in signal transduction analysis and constitute a rapidly growing class of therapeutics for targeted intervention in human diseases. To determine how kinase-selective drugs interfere with cellular physiology on the molecular level, experimental strategies relying on the affinity capture of cellular targets in combination with protein identification by mass spectrometry have been established for a variety of kinase inhibitors. Importantly, these chemical proteomic methods permit the direct analysis of kinase inhibitor selectivity in biological systems and have led to new insights into the cellular modes of action of kinase-selective small molecule antagonists.

Keywords: Abbreviations; CBR1; carbonyl reductase 1; CID; collision-induced; CDK; cyclin-dependent kinase; ESI; electrospray ionisation; EGFR; epidermal growth factor receptor; ERK; extracellular signal-regulated protein kinase; GAK; cyclin G-activated kinase; MALDI; matrix-assisted laser desorption/ionisation; MAPK; mitogen-activated protein kinase; MS; mass spectrometry; RIPK2; receptor-interacting protein kinase 2; RTK; receptor tyrosine kinase; TNF-α; tumour necrosis factor αProtein kinase; Chemical proteomic; Affinity purification; Mass spectrometry; Inhibitor selectivity; Target identification

Coupling phosphoryl transfer and substrate interactions in protein kinases by Scot A. Lieser; Brandon E. Aubol; Lilly Wong; Patricia A. Jennings; Joseph A. Adams (pp. 191-199).

Protein kinases control cell signaling events through the ATP-dependent phosphorylation of serine, threonine and tyrosine residues in protein targets. The recognition of these protein substrates by the kinases relies on two principal factors: proper subcellular co-localization and molecular interactions between the kinase and substrate. In this review, we will focus on the kinetic role of the latter in conveying favorable substrate recognition. Using rapid mixing technologies, we demonstrate that the intrinsic thermodynamic affinities of two protein substrates for their respective kinases (Csk with Src and Sky1p with Npl3) are weak compared to their apparent affinities measured in traditional steady -state kinetic assays (i.e.— K_m< K_d). The source of the high apparent affinities rests in a very fast and highly favorable phosphoryl transfer step that serves as a clamp for substrate recognition. In this mechanism, both Csk and Sky1p utilize this step to draw the substrate toward product, thereby, converting a high K_d into a low K_m. We propose that this one form of substrate recognition employed by protein kinases is advantageous since it simultaneously facilitates high apparent substrate affinity and fast protein turnover.

Keywords: Abbreviations; Cbp; Csk binding protein; Cbp peptide; phosphotyrosine peptide derived from Cbp with sequence ISAMpYSSVMK; Csk; COOH terminal Src kinase; Kemptide; peptide substrate with sequence LRRASLG; Npl3; nuclear protein localization 3; PKA; cAMP-dependent protein kinase; Sky1p; SR protein kinase in yeast; SH2; Src homology 2 domain; SH3; Src homology 3 domain; Src; SFK from Rous Sarcoma virusClamping; Kinase; Kinetics; Phosphorylation; Substrate; Recognition

Substrate specificity of protein kinases and computational prediction of substrates by Boštjan Kobe; Thorsten Kampmann; Jade K. Forwood; Pawel Listwan; Ross I. Brinkworth (pp. 200-209).

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of “peptide specificity�? of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.

Keywords: Abbreviations; AGC; protein kinases A, G, C; AMPK; AMP-activated protein kinase; CaMK; calmodulin-dependent kinase; CDK; cyclin-dependent kinase; CMGC kinases; cyclin-dependent kinases, MAP kinases, glycogen synthase kinase 3 (GSK3) and CK2-related protein kinases; FRET; fluorescence resonance energy transfer; GSK3; glycogen synthase kinase 3; KESTREL; kinase substrate tracking and elucidation; MAP kinase; mitogen-activated protein kinase; PDB; Protein Data Bank; PHK; phosphorylase kinase; PI3 kinase; 3-phosphatidyl inositol kinase; PKA; protein kinase A; PKB; protein kinase B; PKC; protein kinase C; PKG; protein kinase G; PS-OPL; positional scanning of oriented peptide libraries; SDR; specificity-determining residueBioinformatics; Phosphorylation; Protein kinase; Protein structure; Signal transduction; Substrate specificity

Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases by Yuh-Ping Chong; Kim Kui Ia; Terrence D. Mulhern; Heung-Chin Cheng (pp. 210-220).

Src-family kinases (SFKs) are protooncogenic enzymes controlling mammalian cell growth and proliferation. The activity of SFKs is primarily regulated by two tyrosine phosphorylation sites: autophosphorylation of a conserved tyrosine (Y_A) in the kinase domain results in activation while phosphorylation of the regulatory tyrosine (Y_T) near the C-terminus leads to inactivation. The phosphorylated Y_T (pY_T) engages in intramolecular interactions that stabilise the inactive conformation of SFKs. These inhibitory intramolecular interactions include the binding of pY_T to the SH2 domain and the binding of the SH2-kinase linker to the SH3 domain. Thus, SFKs are active upon (i) disruption of the inhibitory intramolecular interactions, (ii) autophosphorylation of Y_A and/or (iii) dephosphorylation of pY_T. Since aberrant activation of SFKs contributes to cancer, SFKs in normal cells are kept inactive by multiple endogenous inhibitors classified as catalytic and non-catalytic inhibitors. The catalytic inhibitors include C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) that phosphorylate Y_T of SFKs, as well as the protein tyrosine phosphatases that dephosphorylate pY_A of the activated SFKs. The non-catalytic inhibitors inactivate SFKs by direct binding. CHK is unique among these inhibitors because it employs both catalytic and non-catalytic mechanisms to inhibit SFKs. Other known non-catalytic inhibitors include WASP, caveolin and RACK1, which function to down-regulate SFKs in specific subcellular locations. This review discusses how the various endogenous SFK inhibitors cooperate to regulate SFKs in normal cells. As chemical compounds that can selectively inhibit SFKs in vivo are potential anti-cancer therapeutics, this review also discusses how investigation into the inhibitory mechanisms of the endogenous inhibitors will benefit the design and screening of these compounds.

Keywords: Abbreviations; SFK; Src-family protein tyrosine kinases; CSK; C-terminal Src kinase; CHK; CSK-homologous kinase; SH2 and SH3 domains; Src-homology 2 and Src-homology 3 domains; Y; _T; C-terminal tail regulatory tyrosine of SFKs; Y; _A; autophosphorylated tyrosine in the activation loop of SFKs; PPII helix; polyproline type II helix; FAK; focal adhesion kinase; HIV-1; human immunodeficiency virus-1; PEP; proline-enriched tyrosine phosphatase; TCPTP; T-cell protein tyrosine phosphatase; SHP1; tandem SH2 domain-containing tyrosine phosphatase-1; SHP2; tandem SH2 domain-containing tyrosine phosphatase-2; Cbp; CSK-binding protein; ErbB2; protein product of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, it is an EGF-receptor-like protein tyrosine kinase; c-Kit; protein product of the cellular homolog of the feline sarcoma viral oncogene v-kit, it is a type 3 transmembrane receptor for stem cell factor, its intracellular portion contains a protein tyrosine kinase domain; HEK 293T cells; human embryonic kidney 293T cells; WASP; Wiskott–Aldrich syndrome protein; RACK1; the receptor of activated protein kinase C (RACK1)Inhibitor; Src-family protein tyrosine kinase; Protein tyrosine phosphatase; Non-catalytic inhibitory mechanism; Cancer

Target flexibility in molecular recognition by J. Andrew McCammon (pp. 221-224).

Induced-fit effects are well known in the binding of small molecules to proteins and other macromolecular targets. Among other targets, protein kinases are particularly flexible proteins, so that such effects should be considered in attempts at structure-based inhibitor design for kinase targets. This paper outlines some recent progress in methods for including target flexibility in computational studies of molecular recognition. A focus is the “relaxed complex method,�? in which ligands are docked to an ensemble of conformations of the target, and the best complexes are re-scored to provide predictions of optimal binding geometries. Early applications of this method have suggested a new approach to the development of inhibitors of HIV-1 Integrase.

Keywords: Structure-based drug discovery; Computer-aided drug design; Induced fit; Molecular dynamics; Computer simulation; Free energy

Accounting for global protein deformability during protein–protein and protein–ligand docking by Andreas May; Martin Zacharias (pp. 225-231).

Computational docking methods are valuable tools aimed to simplify the costly process of drug development and improvement. Most current approaches assume a rigid receptor structure to allow virtual screening of large numbers of possible ligands and putative binding sites on a receptor molecule. However, inclusion of receptor flexibility can be of critical importance since binding of a ligand can lead to changes in the receptor protein conformation that are sterically necessary to accommodate a ligand. Recent approaches to efficiently account for receptor flexibility during docking simulations are reviewed. In particular, accounting efficiently for global conformational changes of the protein backbone during docking is a still challenging unsolved problem. An approximate method has recently been suggested that is based on relaxing the receptor conformation during docking in pre-calculated soft collective degrees of freedom (M. Zacharias, Rapid protein–ligand docking using soft modes from molecular dynamics simulations to account for protein deformability: binding of FK506 to FKBP, Proteins: Struct., Funct., Genet. 54 (2004) 759–767). Test applications on protein–protein docking and on docking the inhibitor staurosporine to the apo-form of cAMP-dependent protein kinase A catalytic domain indicate significant improvement of docking results compared to rigid docking at a very modest computational demand. Accounting for receptor conformational changes in pre-calculated global degrees of freedom might offer a promising route to improve systematic docking screening simulations.

Keywords: Abbreviations; cAMP; cyclic-3′,5′-adenosine monophosphate; MD; molecular dynamics; PC; principal components; PKA; cAMP-dependent protein kinase; RNAse; ribonucleaseLigand–receptor interaction; Approximate normal mode; Receptor flexibility; Domain motion; Induced fit binding; Docking minimization

Dynamical binding of proline-rich peptides to their recognition domains by Wei Gu; Volkhard Helms (pp. 232-238).

Recognition of proline-rich sequences plays an important role for the assembly of multi-protein complexes during the course of eukaryotic signal transduction and is mediated by a set of protein folds that share characteristic features. For many complex systems containing proline-rich sequences, multiple binding modes have been found by theoretical and/or experimental studies. In this review, we discuss the different binding modes as well as the correlated dynamics of the peptides and their recognition domains, and some implications to their biological functions. Furthermore, we give an outlook of the systems in the context of systems biology.

Keywords: Abbreviations; PRS; proline-rich sequences; SH2/SH3; Src homology 2/3; GYF; glycine-tyrosine-phenylalanine; WW; protein domains containing two conserved tryptophans (W) spaced 20–22 amino acids apart; PPI/PPII; poly-proline helix type I/IIPRS (proline-rich sequence); SH3 domain; GYF domain; WW domain; Register shift; Peptide binding

Multiple molecular recognition mechanisms. Cytochrome P450—A case study by Rebecca C. Wade; Domantas Motiejunas; Karin Schleinkofer; Sudarko; Peter J. Winn; Amit Banerjee; Andrei Kariakin; Christiane Jung (pp. 239-244).

Biomolecular recognition is complex. The balance between the different molecular properties that contribute to molecular recognition, such as shape, electrostatics, dynamics and entropy, varies from case to case. This, along with the extent of experimental characterization, influences the choice of appropriate computational approaches to study biomolecular interactions. Here, we present computational studies of cytochrome P450 enzymes and their interactions with small molecules and with other proteins. These interactions exemplify some of the diversity of molecular determinants of binding affinity and specificity observed for proteins and we discuss some of the challenges that they pose for molecular modelling and simulation.

Keywords: Abbreviations; FTIR; Fourier transform infra-red spectroscopy; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; P450; cytochrome P450; Pdx; putidaredoxin; RAMD; random acceleration molecular dynamicsMolecular recognition; Cytochrome P450; Molecular dynamics simulation; Protein dynamics; Protein docking

Development of specific Rho-kinase inhibitors and their clinical application by Masahiro Tamura; Hiroshi Nakao; Hideo Yoshizaki; Masami Shiratsuchi; Hiromichi Shigyo; Hajime Yamada; Takatoshi Ozawa; Junko Totsuka; Hiroyoshi Hidaka (pp. 245-252).

Hexahydro-1-(isoquinoline-5-sulfonyl)-1H-1,4-diazepine, HA-1077, is a known selective inhibitor of Rho-kinase. Although its IC₅₀ value against Rho-kinase is more than 10 times lower than those for kinases such as PKA, PKB, PKC, PKG, MLCK, CaMKII and others, the molecule still retains relative potent inhibition activities against these kinases. In order to produce highly specific Rho-kinase inhibitors, several HA-1077 analogs were synthesized and their kinase inhibition properties evaluated. ( S)-Hexahydro-1-(4-ethenylisoquinoline-5-sulfonyl)-2-methyl-1H-1,4-diazepine was found to be a potent Rho-kinase inhibitor. The IC₅₀ value against Rho-kinase was 6 nM, while those against other kinases remained at almost the same level as that of HA-1077. Furthermore, we designed HA-1077 analogs on the basis of the complex structure of PKA and HA-1077. Amongst these, ( S)-hexahydro-4-glycyl-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1H-1,4-diazepine and other glycine derivatives were found to be highly specific Rho-kinase inhibitors. These Rho-kinase specific inhibitors were applied to rabbit ocular hypertensive models and were shown to reduce intraocular pressure. These results demonstrate that the new 5-isoquinolinesulfonylamides are not only potent ROCK selective compounds, but are also useful compounds for clinical applications.

Keywords: Rho-kinase inhibitor; HA-1077 analog; Intraocular pressure

MAPK signalling pathways as molecular targets for anti-inflammatory therapy—from molecular mechanisms to therapeutic benefits by Bozena Kaminska (pp. 253-262).

Excessive inflammation is becoming accepted as a critical factor in many human diseases, including inflammatory and autoimmune disorders, neurodegenerative conditions, infection, cardiovascular diseases, and cancer. Cerebral ischemia and neurodegenerative diseases are accompanied by a marked inflammatory reaction that is initiated by expression of cytokines, adhesion molecules, and other inflammatory mediators, including prostanoids and nitric oxide. This review discusses recent advances regarding the detrimental effects of inflammation, the regulation of inflammatory signalling pathways in various diseases, and the potential molecular targets for anti-inflammatory therapy. Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases that mediate fundamental biological processes and cellular responses to external stress signals. Increased activity of MAPK, in particular p38 MAPK, and their involvement in the regulation of the synthesis of inflammation mediators at the level of transcription and translation, make them potential targets for anti-inflammatory therapeutics. Inhibitors targeting p38 MAPK and JNK pathways have been developed, and preclinical data suggest that they exhibit anti-inflammatory activity. This review discusses how these novel drugs modulate the activity of the p38 MAPK and JNK signalling cascades, and exhibit anti-inflammatory effects in preclinical disease models, primarily through the inhibition of the expression of inflammatory mediators. Use of MAPK inhibitors emerges as an attractive strategy because they are capable of reducing both the synthesis of pro-inflammatory cytokines and their signalling. Moreover, many of these drugs are small molecules that can be administered orally, and initial results of clinical trials have shown clinical benefits in patients with chronic inflammatory disease.

Keywords: Abbreviations; ARE; AU-rich element; ASK; Apoptosis Signal regulating Kinase; ATF-2; Activating Transcription Factor 2; CRE; Cyclic AMP Responsive Element; ERK; Extracellular signal regulated kinase; iNOS; inducible Nitric Oxide Synthase; JNK; c-Jun N-terminal Kinase; LPS; lipopolysaccharide; MEF-2C; Myocyte enhancer factor 2C; MAPKAP-K2/3; MAP kinase-activated protein kinase 2/3; MEK; MAP/ERK kinase; MKK; MAP kinase kinase; MLK; Mixed lineage kinase; NIK; NF-κB-inducing kinase; TAK1; Transforming growth factor-activated kinase 1; TNF; Tumor necrosis factor; TPL2; Tumor progression locus 2Inflammatory disease; Neuroinflammation; Pro-inflammatory cytokine; MAP kinase; Transcription factor; Immunosuppressant; Inhibitor

Features and potentials of ATP-site directed CK2 inhibitors by Stefania Sarno; Mauro Salvi; Roberto Battistutta; Giuseppe Zanotti; Lorenzo A. Pinna (pp. 263-270).

A panel of quite specific, fairly potent and cell-permeable inhibitors of protein kinase CK2 belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines have been developed, with K_i values in the submicromolar range. Nine structures have been solved to date of complexes between the catalytic α subunit of CK2 and a number of these compounds, many of which display a remarkable specificity toward CK2 as compared to a panel of >30 kinases tested. The structural basis for such selectivity appears to reside in the shape and size of a hydrophobic pocket adjacent to the ATP binding site where these ATP competitive ligands are entrapped mainly by van der Waals interactions and by an energy contribution derived from the hydrophobic effect. In CK2, this cavity is smaller than in the majority of other protein kinases due to a number of unique bulky apolar residues. Consequently, the replacement of two of these residues (V66 and I174) in human CK2 alpha with alanines gives rise to mutants, which are markedly less susceptible than wild type to these classes of inhibitors. Cell-permeable CK2 inhibitors have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signalling pathways affected by CK2 and/or to validate the identification of in vivo targets of this pleiotropic kinase. Moreover, the remarkable pro-apoptotic efficacy of these compounds toward cell lines derived from a wide spectrum of tumors, disclose the possibility that in perspective CK2 inhibitors might become leads for the development of anti-cancer drugs.

Keywords: Abbreviations; CK2; casein kinase 2; Dyrk1A; dual specificity tyrosine phosphorylation-regulated kinase; DMAT; 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole; GSK3; glycogen synthase kinase 3; HSP90; heat shock protein; MNX; 1,8-dihydroxy-4-nitro-xanthen-9-one; PI3 kinase; phosphoinositide-3 kinaseProtein kinase CK2; Casein kinase-2; Protein kinase inhibitor; Apoptosis; Neoplasia

Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: Evaluation of their effects on cells and different molecular forms of human CK2 by Piotr Zien; James S. Duncan; Janusz Skierski; Maria Bretner; David W. Litchfield; David Shugar (pp. 271-280).

The development of selective cell-permeable inhibitors of protein kinase CK2 has represented an important advance in the field. However, it is important to not overlook the existence of discrete molecular forms of CK2 that arise from the presence of distinct isozymic forms, and the existence of the catalytic CK2 subunits as free subunits and in complexes with the regulatory CK2β subunits and, possibly, other proteins. This review examines two recently developed, and presently widely applied, CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBBt) and the related 4,5,6,7-tetrabromobenzimidazole (TBBz), the latter of which was previously shown to discriminate between different molecular forms of CK2 in yeast. We have shown, by spectrophotometric titration, that TBBt, with a p K_a ≈5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a p K_a ≈9, is predominantly (>95%) in the neutral form, both of obvious relevance to their modes of binding. In vitro, TBBt inhibits different forms of CK2 with K_i values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with K_i values ranging from 70 to 510 nM. Despite their general similar in vitro activities, TBBz is more effective than TBBt in inducing apoptosis and, to a lesser degree, necrosis, in transformed human cell lines. Finally, development of shRNA strategies for the selective knockdown of the CK2α and CK2α′ isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation.

Keywords: Abbreviations; CK2; protein kinase CK2; TBBt; tetrabromobenzotriazole; TBBz; tetrabromobenzimidazole; DRB; 5,6-dichloro-1-(β-; d; -ribofuranosyl)benzimidazole; CKA1 and CKA2; the catalytic subunits of protein kinase CK2 in; S. cerevisiae; FACS; fluorescence activated cell sorting; C9; the C9 component of the complement system; CPα; the α subunit of the heterodimeric actin capping protein; La; RNA-binding protein LaProtein kinase CK2; Isoform; Selective inhibitor; Tetrabromobenzotriazole; Tetrabromobenzimidazole; Apoptosis; shRNA knockdown

Mammalian histidine kinases by Paul G. Besant; Paul V. Attwood (pp. 281-290).

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.

Keywords: Abbreviations; NDPK; nucleoside diphosphate kinase; NTP; nucleoside triphosphate; HHK; histone H4 histidine kinase; K; _I; inhibitory constant; BCKDHK; branched chain α-ketoacid dehydrogenase kinase; PDHK; pyruvate dehydrogenase kinase; eEF-2; eukaryotic elongation factor-2; eEF-2 kinase; eukaryotic elongation factor-2 kinase; RPTLC; reverse-phase thin layer chromatography; k; _cat; catalytic rate constant; K; _m; Michaelis constant; LHPPase; lysine/histidine protein phosphataseHistidine kinase; Phosphohistidine; NDP kinase; Histone H4; Protein histidine phosphatase; Kinase inhibitor

Reversible phosphorylation of histidine residues in vertebrate proteins by Susanne Klumpp; Josef Krieglstein (pp. 291-295).

Knowledge on kinases and phosphatases acting on serine, threonine and tyrosine residues of vertebrate proteins is huge. These enzymes are still under intensive investigation at present. This is in sharp contrast to what is known about kinases and phosphatases acting on histidine, arginine, lysine and aspartate residues in vertebrate proteins. It also is in contrast to extensive studies of histidine/aspartate phosphorylation in prokaryotes. This minireview briefly summarizes what we have learned about the reversible phosphorylation of histidine residues in mammals. It is described how the field developed during 40 years of science. The article especially highlights the discovery of the first protein histidine phosphatase from vertebrates. Having identified and characterized a protein histidine phosphatase provides at least one desperately required tool to handle and study phosphorylation and dephosphorylation of histidine residues in vertebrates in more detail. Recent evidence even suggests an involvement of histidine phosphorylation in signal transduction.

Keywords: Abbreviations; ACL; ATP-citrate lyase; PHP; protein histidine phosphatase; NDPKs; nucleoside diphosphate kinasesProtein phosphorylation; Protein histidine phosphatase; Protein histidine kinase; ATP-citrate lyase; G-protein β-subunit; Nucleoside diphosphate kinase

Targeting protein kinase C and “non-kinase�? phorbol ester receptors: Emerging concepts and therapeutic implications by Marcelo G. Kazanietz (pp. 296-304).

Phorbol esters, natural compounds that mimic the action of the lipid second messenger diacylglycerol (DAG), are known to exert their biological actions through the activation of classical and novel protein kinase C (PKC) isozymes. Phorbol esters, via binding to the PKC C1 domains, cause major effects on mitogenesis by controlling the activity of cyclin–cdk complexes and the expression of cdk inhibitors. In the last years it became clear that phorbol esters activate other molecules having a C1 domain in addition to PKCs. One of the most interesting families of “non-kinase�? phorbol ester receptors is represented by the chimaerins, lipid-regulated Rac-GAPs that modulate actin cytoskeleton reorganization, migration, and proliferation. The discovery of the chimaerins and other “non-kinase�? phorbol ester receptors has major implications in the design of agents for cancer therapy.

Keywords: Abbreviations; DAG; diaclyclycerol; DGK; diacylglycerol kinase; ILV; indolactam V; FRET; Florescence Resonance Energy Transfer; PAH; polycyclic aromatic hydrocarbon; PDBu; phorbol 12, 13-dibutyrate; PDK1; phosphatidylinositide-dependent kinase 1; PKC; protein kinase C; PLC; phospholipase; PMA or TPA; phorbol 12-myristate 13-acetate or phorbol 12-tetradecanoyl 13-acetate; RACK; Receptor for activated C kinasePKC; Chimaerin; C1 domain; Phorbol ester; Diacylglycerol; Bryostatin

The role of SMG-1 in nonsense-mediated mRNA decay by Akio Yamashita; Isao Kashima; Shigeo Ohno (pp. 305-315).

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.

Keywords: Abbreviations; NMD; nonsense-mediated mRNA decay; EJC; exon–junction complex; PTC; premature translation termination codon; IC; initial SMG-1–Upf1 complex; PIKK; phosphoinositide 3-kinase related kinase; ATM; ataxia telangiectasia mutated; ATR; ATM and Rad3 related; mTOR; target of rapamycin; DNA-PK; DNA-dependent protein kinase; DSE; downstream sequence elements; PABP; poly(A) binding proteinNMD; PTC; PIKK; SMG-1; Phosphorylation; Dephosphorylation

Quantifying ERK2–protein interactions by fluorescence anisotropy: PEA-15 inhibits ERK2 by blocking the binding of DEJL domains by Kari Callaway; Mark A. Rainey; Kevin N. Dalby (pp. 316-323).

While mitogen-activated protein kinase signaling pathways constitute highly regulated networks of protein–protein interactions, little quantitative information for these interactions is available. Here we highlight recent fluorescence anisotropy binding studies that focus on the interactions of ERK1 and ERK2 with PEA-15 (antiapoptotic phosphoprotein enriched in astrocytes—15 kDa), a small protein that sequesters ERK2 in the cytoplasm. The regulation of ERK2 by PEA-15 is appraised in the light of a simple equilibrium-binding model for reversible ERK2 nucleoplasmic–cytoplasmic shuttling, which elaborates on the theory of Burack and Shaw (J. Biol. Chem. 280, 3832–3837; 2005). Also highlighted is the recent observation that the peptide N-Q KG KPRD LE LPLSPSL-C, derived from the docking site for ERK/JNK and LEL (DEJL) in Elk-1, displaces PEA-15 from ERK2. It is proposed that the C-terminus of PEA-15 (¹²¹ LX LXXXX KK¹²⁹) is a reverse DEJL domain [which has a general consensus of R/K-ϕ_A-X_3/4-ϕ_B, where ϕ_A and ϕ_B are hydrophobic residues (Leu, Ile, or Val)], which mediates one arm of a bidentate PEA-15 interaction with ERK2. The notion that PEA-15 is a potent inhibitor of many ERK2-mediated phosphorylations, by virtue of its ability to block ERK2–DEJL domain interactions, is proposed.

Keywords: Abbreviations; DEF; docking site for ERK and FXFP; DEJL; docking site for ERK/JNK and LEL; ERK1/2; extracellular signal-regulated protein kinases 1 and 2; EtsΔ138; murine (His; ₆; -tagged) Ets; ^1–138; PEA-15; phosphoprotein enriched in astrocytes—15 kDa; PEA-15*; PEA-15 covalently labeled with fluorescein; Hepes; N; -(2-hydroxyethyl)piperazine-; N; ′-2-ethanesulfonic acid; MAPK; mitogen-activated protein kinase; MAPKK1/2; mitogen-activated protein kinase kinases 1 and 2; CFP; cyan fluorescent protein; YFP; yellow fluorescent protein; FRET; fluorescence resonance energy transfer; FADD; Fas-associating protein with death domain; EGF; epidermal growth factor; PMA; phorbol myristate acetateMAPK kinase; ERK2; Fluorescence anisotropy; Nucleoplasmic; Cytoplasmic:Shuttling

Alternative splicing: A new drug target of the post-genome era by Masatoshi Hagiwara (pp. 324-331).

Alternative splicing allows for the creation of multiple distinct mRNA transcripts from a given gene in a multicellular organism. Pre-mRNA splicing is catalyzed by a multi-molecular complex, including serine/arginine-rich (SR) proteins, which are highly phosphorylated in living cells, and thought to play crucial roles in spliceosomal formation and in the regulation of alternative splicing. Recently, reports of low molecular compounds, which alter splicing pattern of genes, have been accumulated. A benzothiazole compound TG003, a kinase inhibitor that targets Clk1 and Clk4, suppressed dissociation of nuclear speckles, altered the splicing patterns, and rescued the embryonic defects induced by excessive Clk activity. The emerging inhibitors of the signal transduction pathways regulating pre-mRNA alternative splicing may open the way to therapies against diseases caused by missplicing.

Keywords: Abbreviations; SR; serine/arginine-rich; hnRNP; heterogeneous nuclear ribonucleoparticle; ESE; exonic splicing enhancer; ISE; intronic splicing enhancer; ESS; exonic splicing silencer; ISS; intronic splicing silencer; PKC; protein kinase C; PI 3-kinase; phosphatidylinositol 3-kinase; SAM68; SRC-associated in mitosis, 68 kDa; STAR; signal transduction and activation of RNA; ERK; extracellular signal-regulated kinase; STREX; stress axis; CaMKIV; Ca; ²⁺; /calmodulin-dependent protein kinase IV; CaRRE; CaMKIV-responsive RNA element; NSSR; neural-salient serine/arginine-rich protein; ICH-1; interleukin-1β converting enzyme homologue 1; SRPK; SR protein kinase; Clk; Cdc2-like kinase; PRP4; pre-mRNA processing mutant 4; DRB; 5,6-dichloro-1-β-; d; -ribo-furanosylbenzimidazole; PP1; protein phosphatase 1; PP2A; protein phosphatase 2A; SMN; survival of motor neuron A; lternative splicing; SR protein; Clk; SRPK; TG003

Proteins and Proteomics Cumulative Contents (pp. 332-333).

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