The split-hybrid system. Uncoding multiprotein networks and defining mutations that affect protein interactions.
Explore the source record for details and available documents.
Bacterial Proteins↗
Biomedical subjects
M F Hoekstra
Publications and source records attributed to M F Hoekstra.
Crystal structure of a conformation-selective casein kinase-1 inhibitor.
Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.
Casein Kinases↗
The yeast split-hybrid system.
Explore the source record for details and available documents.
Bacterial Proteins↗
Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.
The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.
Adaptor Proteins, Signal Transducing↗
A new molecular link between the fibrillar and granulovacuolar lesions of Alzheimer's disease.
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder involving select neurons of the hippocampus, neocortex, and other regions of the brain. Markers of end stage disease include fibrillar lesions, which accumulate hyperphosphorylated tau protein polymerized into filaments, and granulovacuolar lesions, which appear primarily within the hippocampus. The mechanism by which only select populations of neurons develop these lesions as well as the relationship between them is unknown. To address these questions, we have turned to AD tissue to search for enzymes specifically involved in tau hyperphosphorylation. Recently, we showed that the principal phosphotransferases associated with AD brain-derived tau filaments are members of the casein kinase-1 (CK1) family of protein kinases. Here we report the distribution of three CK1 isoforms (Ckialpha, Ckidelta, and Ckiepsilon) in AD and control brains using immunohistochemistry and Western analysis. In addition to colocalizing with elements of the fibrillar pathology, CK1 is found within the matrix of granulovacuolar degeneration bodies. Furthermore, levels of all CK1 isoforms are elevated in the CA1 region of AD hippocampus relative to controls, with one isoform, Ckidelta, being elevated >30-fold. We propose that overexpression of this protein kinase family plays a key role in the hyperphosphorylation of tau and in the formation of AD-related pathology.
Aged↗
Genetic pathway to recurrent chromosome translocations in murine lymphoma involves V(D)J recombinase.
Chromosome translocations involving antigen receptor loci are a genetic hallmark of non-Hodgkin's lymphomas in humans. Most commonly, these translocations result in juxtaposition of the immunoglobulin heavy-chain (IgH) locus with one of several cellular proto-oncogenes, leading to deregulated oncogene expression. The V(D)J recombinase, which mediates physiologic rearrangements of antigen receptor genes, may play a mechanistic role in some lymphoma translocations, although evidence is indirect. A high incidence of B-lineage lymphomas has been observed in mice with severe combined immunodeficiency (SCID) and p53-null mutations. We show that these tumors are characteristic of the pro-B-cell stage of development and that they harbor recurrent translocations involving chromosomes 12 and 15. Fluorescence in situ hybridization (FISH) shows retention of IgH sequences on the derivative chromosome 12, implying that breakpoints involve the IgH locus. Pro-B-cell lymphomas were suppressed in SCID p53(-/-) mice by a Rag-2-null mutation, demonstrating that DNA breaks generated during V(D)J recombination are required for oncogenic transformation, and suggesting that t(12;15) arise during attempted IgH rearrangement in pro-B cells. These studies indicate that the oncogenic potential inherent in antigen receptor diversification is controlled in vivo by efficient rejoining of DNA ends generated during V(D)J recombination and an intact cellular response to DNA damage.
Animals↗
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.
UNLABELLED: Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. KEYWORDS: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3
Adenosine Triphosphatases↗
AKAP79 inhibits calcineurin through a site distinct from the immunophilin-binding region.
Targeting of protein kinases and phosphatases provides additional specificity to substrate selectivity in cellular signaling. In the case of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, AKAP79 has been shown to bind calcineurin and inhibit its activity in vitro (Coghlan, V., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science 267, 108-111). In the present study, we characterized the binding regions on calcineurin A (CnA) and AKAP79 that are important for this interaction. Residues 30-98 and 311-336 on CnA, and residues 108-280 on AKAP79 were found to be important for binding. The binding of CnA by AKAP79 does not require the calcineurin B subunit, and occurs in a region distinct from where the immunosuppressant-immunophilin complex bind. AKAP79 also bound to CnA in cells transfected with AKAP79 and CnA. To determine the function of AKAP79-calcineurin interaction in intact cells, we measured the dephosphorylation and subsequent activation of NFAT, a transcription factor that is a substrate for calcineurin. Overexpression of AKAP79 inhibited NFAT dephosphorylation, resulting in a decrease in NFAT activation. These results demonstrated that AKAP79 can bind to and inhibit calcineurin activity in vivo, suggesting a physiological role for AKAP79-calcineurin interaction in NFAT-mediated signaling.
Binding Sites↗
Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control.
In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.
Ataxia Telangiectasia Mutated Proteins↗
Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase.
The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.
DNA Damage↗
Changes in protein composition of meiotic nodules during mammalian meiosis.
Homologous chromosome synapsis and meiotic recombination are facilitated by several meiosis-specific structures: the synaptonemal complex (SC), and two types of meiotic nodules: (1) early meiotic nodules (MNs), also called zygotene nodules or early recombination nodules, and (2) late recombination nodules (RNs). The former are thought to be nucleoprotein complexes involved in the check for homology preceding, or accompanying synapsis, while the latter have been shown to be involved in reciprocal recombination. We have examined by immunocytochemistry the meiotic localization of a series of proteins at sites along the asynapsed axial elements prior to homologous synapsis and at sites along the SCs following synapsis. Several of the proteins examined have been implicated in repair/recombination and include RAD51, a mammalian homolog of the Escherichia coli RecA protein; Replication Protein-A (RPA), a single-strand DNA binding protein; and MLH1, a mismatch repair protein which is a homolog of the E. coli MutL protein. In addition two proteins were examined that have been implicated in meiotic checkpoints: ATM, the protein mutated in the human disease Ataxia Telangiectasia, and ATR, another member of the same family of PIK kinases. We present evidence that these proteins are all components of meiotic nodules and document changes in protein composition of these structures during zygonema and pachynema of meiotic prophase in mouse spermatocytes. These studies support the supposition that a subset of MNs are converted into RNs. However, our data also demonstrate changes in protein composition within the context of early MNs, suggesting a differentiation of these nodules during the process of synapsis. The same changes in protein composition occurred on both the normal X axis, which has no homologous pairing partner in spermatocytes, and on the axes of aberrant chromosomes that nonhomologously synapse during synaptic adjustment. These findings suggest that DNA sequences associated with MNs still must undergo an obligatory processing, even in the absence of interactions between homologous chromosomes.
Animals↗
Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.
BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.
Amino Acid Sequence↗
The beta-adrenergic receptor kinase interacts with the amino terminus of the G protein beta subunit.
Desensitization of G protein-coupled receptors involves phosphorylation of the receptors by G protein-coupled receptor kinases, such as the beta-adrenergic receptor kinase (beta ARK). beta ARK activity depends upon its translocation from the cytoplasm to the membrane. The beta gamma subunits of G proteins bind to beta ARK and recruit the kinase to the membrane. The G beta gamma binding domain is localized to a carboxyl terminal region of beta ARK but the beta ARK binding domain of G beta gamma is not known. We used the yeast two-hybrid assay to characterize the interaction between G beta and beta ARK. We demonstrate an interaction between the carboxyl terminus of beta ARK and G beta 2. The strength of this interaction is increased when the VP16 transactivation domain is placed on the carboxyl end of G beta 2, indicating that an accessible G beta 2 amino terminus is important for its interaction with beta ARK. In addition, we show that amino acids 1 to 145 of G beta 2 are sufficient for beta ARK binding.
Amino Acid Sequence↗
p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs.
The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.
Animals↗
p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes.
We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta. In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.
Amino Acid Sequence↗
Responses to DNA damage and regulation of cell cycle checkpoints by the ATM protein kinase family.
In mammalian cells, four protein kinases form the PI3-kinase-related protein kinase (PIK) superfamily. These four enzymes-FRAP, DNA-PK, ATM, and ATR-are distinguished by their large size (all are >2500 amino acids), their common primary sequence relatedness through the carboxy-terminal protein kinase domain, and their sequence similarity to the p110 lipid kinase subunit of PI3-kinase. FRAP (FKBP12 and rapamycin-binding protein kinase) participates in mitogenic and growth factor responses in G1 and may regulate specific mRNA translation signals. DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad 3 related) are thought to participate in responses to nuclear cues that activate DNA rearrangements or cell cycle arrests. Recent studies in this protein kinase family indicate an important role for ATM and ATR in a meiotic surveillance mechanism that may regulate proper chromosome transmission.
Adenosine Triphosphatases↗
ATM and RPA in meiotic chromosome synapsis and recombination.
ATM is a member of the phosphatidylinositol 3-kinase (PIK)-like kinases, some of which are active in regulating DNA damage-induced mitotic cell-cycle checkpoints. ATM also plays a role in meiosis. Spermatogenesis in Atm-/- male mice is disrupted, with chromosome fragmentation leading to meiotic arrest; in human patients with ataxia-telangiectasia (A-T), gonadal atrophy is common. Immuno-localization studies indicate that ATM is associated with sites along the synaptonemal complex (SC), the specialized structure along which meiotic recombination occurs. Recombination, preceded by pairing of homologous chromosomes, is thought to require heteroduplex formation between homologous DNA, followed by strand exchange. These early meiotic steps (entailing the formation and processing of meiotic recombination intermediates with DNA-strand interruptions) require ssDNA-binding proteins such as replication protein A (RPA; refs 5-7). In somatic cells, DNA damage induces ATM-dependent phosphorylation of RPA. We demonstrate here that ATM and RPA co-localize along synapsed meiotic chromosomes and at sites where interactions between ectopic homologous chromosome regions appear to initiate. In Atm-/- meiotic prophase spermatocytes, immuno-localization shows that RPA is present along synapsing chromosomes and at sites of fragmentation of the SC. These results suggest that RPA and ATM co-localize at sites where interhomologous-DNA interactions occur during meiotic prophase and where breaks associated with meiotic recombination take place after synapsis, implying a possible functional interaction between these two proteins.
Animals↗
Casein kinase 1 is tightly associated with paired-helical filaments isolated from Alzheimer's disease brain.
The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of approximately 2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the alpha branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.
Adult↗