Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.
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PMID:The FHA domain is a modular phosphopeptide recognition motif. 1051 19

It was proposed previously that the FHA2 domain of the yeast protein kinase Rad53 has dual specificity toward pY and pT peptides. The consensus sequences of pY peptides for binding to FHA2, as well as the solution structures of free FHA2 and FHA2 complex with a pY peptide derived from Rad9, have been obtained previously. We now report the use of a pT library to screen for binding of pT peptides with the FHA2 domain. The results show that FHA2 binds favorably to pT peptides with Ile at the +3 position. We then searched the Rad9 sequences with a pTXXI/L motif, and tested the binding affinity of FHA2 toward ten pT peptides derived from Rad9. One of the peptides, (599)EVEL(pT)QELP(607), displayed the best binding affinity (K(d)=12.9 microM) and the greatest chemical shift changes. The structure of the FHA2 complex with this peptide was then determined by solution NMR and the structure of the complex between FHA2 and the pY peptide (826)EDI(pY)YLD(832) was further refined. Structural comparison of these two complexes indicates that the Leu residue at the +3 position in the pT peptide and that at the +2 position in the pY peptide occupy a very similar position relative to the binding site residues from FHA2. This can explain why FHA2 is able to bind both pT and pY peptides. This position change from +3 to +2 could be the consequence of the size difference between Thr and Tyr. Further insight into the structural basis of ligand specificity of FHA domains was obtained by comparing the structures of the FHA2-pTXXL complex obtained in this work and the FHA1-pTXXD complex reported in the accompanying paper.
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PMID:Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes. 1184 68

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.
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PMID:cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts. 1245 70

Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.
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PMID:Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling. 1291 50

Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mek1 kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase. We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.
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PMID:Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic double strand break repair in budding yeast. 1459 9

Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF.
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PMID:Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis. 1513 25

The DNA replication checkpoint is a complex signal transduction pathway, present in all eukaryotic cells, that functions to maintain genomic integrity and cell viability when DNA replication is perturbed. In Schizosaccharomyces pombe the major effector of the replication checkpoint is the protein kinase Cds1. Activation of Cds1 is known to require the upstream kinase Rad3 and the mediator Mrc1, but the biochemical mechanism of activation is not well understood. We report that the replication checkpoint is activated in two stages. In the first stage, Mrc1 recruits Cds1 to stalled replication forks by interactions between the FHA domain of Cds1 and specific phosphorylated Rad3 consensus sites in Mrc1. Cds1 is then primed for activation by Rad3-dependent phosphorylation. In the second stage, primed Cds1 molecules dimerize via phospho-specific interactions mediated by the FHA domains and are activated by autophosphorylation. This two-stage activation mechanism for the replication checkpoint allows for rapid activation with a high signal-to-noise ratio.
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PMID:Two-stage mechanism for activation of the DNA replication checkpoint kinase Cds1 in fission yeast. 1661 6

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.
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PMID:Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery. 1678 25

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.
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PMID:Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog. 1776 19

Mammalian cells respond to DNA double-strand breaks (DSBs) by recruiting DNA repair and cell-cycle checkpoint proteins to such sites. Central to these DNA damage response (DDR) events is the DNA damage mediator protein MDC1. MDC1 interacts with several DDR proteins, including the MRE11-RAD50-NBS1 (MRN) complex. Here, we show that MDC1 is phosphorylated on a cluster of conserved repeat motifs by casein kinase 2 (CK2). Moreover, we establish that this phosphorylation of MDC1 promotes direct, phosphorylation-dependent interactions with NBS1 in a manner that requires the closely apposed FHA and twin BRCT domains in the amino terminus of NBS1. Finally, we show that these CK2-targeted motifs in MDC1 are required to mediate NBS1 association with chromatin-flanking sites of unrepaired DSBs. These findings provide a molecular explanation for the MDC1-MRN interaction and yield insights into how MDC1 coordinates the focal assembly and activation of several DDR factors in response to DNA damage.
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PMID:Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage. 1858 88


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