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Tanya T Paull - One of the best experts on this subject based on the ideXlab platform.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:Abstract The resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs Autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs Autophosphorylation, and also inhibits DNA Ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs Autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs Autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
Susan P Leesmiller - One of the best experts on this subject based on the ideXlab platform.
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Autophosphorylation of ataxia telangiectasia mutated is regulated by protein phosphatase 2a
The EMBO Journal, 2004Co-Authors: Aaron A. Goodarzi, Jyoti Jonnalagadda, David B Young, Susan P Leesmiller, Greg B. G. Moorhead, Ruiqiong Ye, Pauline Douglas, Kum Kum KhannaAbstract:Ionizing radiation induces Autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces Autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce -H2AX foci, suggesting that it induces ATM Autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM Autophosphorylation and activity in vivo.
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non homologous end joining requires that the dna pk complex undergo an Autophosphorylation dependent rearrangement at dna ends
Journal of Biological Chemistry, 2004Co-Authors: Yeturu V R Reddy, Susan P Leesmiller, Qi Ding, Katheryn Meek, Dale A RamsdenAbstract:Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo Autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs Autophosphorylation is sufficient to explain the requirement for kinase activity, in part because Autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE Autophosphorylation sites mutated to alanine allows access to ends through Autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate.
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Autophosphorylation of the catalytic subunit of the dna dependent protein kinase is required for efficient end processing during dna double strand break repair
Molecular and Cellular Biology, 2003Co-Authors: Qi Ding, Susan P Leesmiller, Pauline Douglas, Yeturu V R Reddy, Dale A Ramsden, Wei Wang, Timothy Woods, Katheryn MeekAbstract:The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes Autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that Autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven Autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that Autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, Autophosphorylation at these sites may not be sufficient for kinase disassembly.
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the dna dependent protein kinase is inactivated by Autophosphorylation of the catalytic subunit
Journal of Biological Chemistry, 1996Co-Authors: Doug W Chan, Susan P LeesmillerAbstract:The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes Autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to Autophosphorylation of DNA-PKcs. Our data also suggest that Autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that Autophosphorylation is an important mechanism for the regulation of DNA-PK activity.
Roger J Colbran - One of the best experts on this subject based on the ideXlab platform.
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substrate selective and calcium independent activation of camkii by α actinin
Journal of Biological Chemistry, 2012Co-Authors: Nidhi Jalansakrikar, Ryan K Bartlett, Anthony J Baucum, Roger J ColbranAbstract:Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII Autophosphorylation at Thr-306, but not by Autophosphorylation at Thr-305, whereas Autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.
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differential modulation of ca2 calmodulin dependent protein kinase ii activity by regulated interactions with n methyl d aspartate receptor nr2b subunits and α actinin
Journal of Biological Chemistry, 2005Co-Authors: Alfred J Robison, Ryan K Bartlett, Martha A Bass, Roger J ColbranAbstract:Abstract Neuronal Ca2+/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and α-actinin bind comparable, approximately stoichiometric amounts of Thr286-autophosphorylated CaMKIIα, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr286-autophosphorylated CaMKII bind to α-actinin with similar efficacy, but Autophosphorylation at Thr305/306 or Ca2+/calmodulin binding significantly reduce this binding. Moreover, α-actinin antagonizes CaMKII activation by Ca2+/calmodulin, as assessed by Autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr286 Autophosphorylation and is unaffected by Ca2+-independent Autophosphorylation or Ca2+/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIα with NR2B requires either Thr286 Autophosphorylation or the binding of both Ca2+/calmodulin and adenine nucleotides. NR2B inhibits both the Ca2+/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.
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Autophosphorylation dependent targeting of calcium calmodulin dependent protein kinase ii by the nr2b subunit of then methyl d aspartate receptor
Journal of Biological Chemistry, 1998Co-Authors: Stefan Strack, Roger J ColbranAbstract:Abstract Activation and Thr286Autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx viaN-methyl-d-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for Autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+influx, and Thr286 Autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.
Yi Zhou - One of the best experts on this subject based on the ideXlab platform.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:Abstract The resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs Autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs Autophosphorylation, and also inhibits DNA Ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice.
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dna dependent protein kinase regulates dna end resection in concert with mre11 rad50 nbs1 mrn and ataxia telangiectasia mutated atm
Journal of Biological Chemistry, 2013Co-Authors: Yi Zhou, Tanya T PaullAbstract:The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs Autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs Autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
Jolinda A Traugh - One of the best experts on this subject based on the ideXlab platform.
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analysis of conformational changes during activation of protein kinase pak2 by amide hydrogen deuterium exchange
Journal of Biological Chemistry, 2008Co-Authors: Yuanhao Hsu, David A Johnson, Jolinda A TraughAbstract:During apoptotic stress, protein kinase Pak2 is cleaved by caspase 3 to form a heterotetramer that is constitutively activated following Autophosphorylation. The active protein kinase migrates slightly slower than the inactive holoenzyme when analyzed by gel filtration, suggesting an expanded conformation. Activation of Pak2 comprises a series of structural changes resulting from caspase cleavage, ATP binding, and Autophosphorylation of Pak2. Changes at each step were individually analyzed by amide hydrogen/deuterium exchange coupled with mass spectrometry and compared with inactive Pak2. The auto-inhibited form was shown to bind ATP in the active site, with minor changes in the glycine loop and the autoinhibitory domain (AID). Caspase cleavage produced significant changes in solvent accessibility in the AID and upper lobe of the catalytic domain. Cleavage of ATP-bound Pak2 relaxes the allosteric inhibition, as shown by increased solvent accessibility in the upper and lower lobes, including the G-helix, facilitating the Autophosphorylation of two sites required for activation, Ser-141 in the regulatory domain and Thr-402 in the catalytic domain. Autophosphorylation increased the amide hydrogen/deuterium exchange solvent accessibility of the contact region between the AID and the G-helix, the E-F loop, and the N terminus. Thus, activation of Pak2 via caspase cleavage is associated with structural relaxation of Pak2 that allows for complete auto-phosphorylation, resulting in a more comprehensive solvent-exposed and conformationally dynamic enzyme.
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multisite Autophosphorylation of p21 activated protein kinase γ pak as a function of activation
Journal of Biological Chemistry, 1999Co-Authors: Andrea Gatti, Zhongdong Huang, Polygena T Tuazon, Jolinda A TraughAbstract:p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent Autophosphorylation. Focusing on the ubiquitous γ-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant γ-PAK, while native γ-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of γ-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant γ-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated Autophosphorylation, the two-dimensional profiles of native and recombinant γ-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, γ-PAK Autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight Autophosphorylation sites were identified in Cdc42-activated γ-PAK, six of which are in common with those previously reported in α-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the γ-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with γ-PAK activation.
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cleavage and activation of p21 activated protein kinase γ pak by cpp32 caspase 3 effects of Autophosphorylation on activity
Journal of Biological Chemistry, 1998Co-Authors: Barbara N Walter, Zhongdong Huang, Rolf Jakobi, Polygena T Tuazon, Emad S Alnemri, Gerald Litwack, Jolinda A TraughAbstract:p21-activated protein kinase γ-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant γ-PAK into two peptides; 1–212 contains the majority of the regulatory domain whereas 213–524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [γ-32P]ATP. Peptide 1–212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following Autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after Autophosphorylation on threonine. Following caspase cleavage, a significant lag (∼5 min) is observed before Autophosphorylation and activity are detected. When γ-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After Autophosphorylation of γ-PAK in the presence of Cdc42(GTPγS) or histone 4, both cleavage products contain phosphate and γ-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces Autophosphorylation and protein kinase activity following cleavage. Thus activation of γ-PAK via cleavage by CPP32 is a two-step mechanism wherein Autophosphorylation of the regulatory domain is a priming step, and activation coincides with Autophosphorylation of the catalytic domain.