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Activation Loop (activation + loop)
Selected AbstractsActivation loop 3 and the 170 loop interact in the active conformation of coagulation factor VIIaFEBS JOURNAL, Issue 11 2009Egon Persson The initiation of blood coagulation involves tissue factor (TF)-induced allosteric activation of factor VIIa (FVIIa), which circulates in a zymogen-like state. In addition, the (most) active conformation of FVIIa presumably relies on a number of intramolecular interactions. We have characterized the role of Gly372(223) in FVIIa, which is the sole residue in activation loop 3 that is capable of forming backbone hydrogen bonds with the unusually long 170 loop and with activation loop 2, by studying the effects of replacement with Ala [G372(223)A]. G372A-FVIIa, both in the free and TF-bound form, exhibited reduced cleavage of factor X (FX) and of peptidyl substrates, and had increased Km values compared with wild-type FVIIa. Inhibition of G372A-FVIIa·sTF by p -aminobenzamidine was characterized by a seven-fold higher Ki than obtained with FVIIa·sTF. Crystallographic and modelling data suggest that the most active conformation of FVIIa depends on the backbone hydrogen bond between Gly372(223) and Arg315(170C) in the 170 loop. Despite the reduced activity and inhibitor susceptibility, native and active site-inhibited G372A-FVIIa bound sTF with the same affinity as the corresponding forms of FVIIa, and burial of the N-terminus of the protease domain increased similarly upon sTF binding to G372A-FVIIa and FVIIa. Thus Gly372(223) in FVIIa appears to play a critical role in maturation of the S1 pocket and adjacent subsites, but does not appear to be of importance for TF binding and the ensuing allostery. [source] Fused protein of ,PKC activation loop and PDK1-interacting fragment (,AL-PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cellsGENES TO CELLS, Issue 9 2006Takahiro Seki To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as ,AL-PIF, and characterized its properties in cultured cells. ,AL-PIF consists of two fused proteins of the protein kinase C, (,PKC) activation loop (,AL) and PDK1-interacting fragment (PIF). The phosphorylation of ,AL-PIF was detected with anti-,PKC phospho-Thr505 -specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing ,AL-PIF, basal phosphorylation level at the activation loop of PKB,, ,PKC and ,PKC was reduced, compared with that in control cells, suggesting that ,AL-PIF functions as an inhibitory molecule for PDK1. ,AL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of ,AL-PIF on ,PKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing ,AL-PIF for 3 days. These findings revealed that ,AL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy. [source] The second phase activation of protein kinase C , at late G1 is required for DNA synthesis in serum-induced cell cycle progressionGENES TO CELLS, Issue 4 2003Koichi Kitamura Background: Cell lines that stably over-express protein kinase C (PKC) , frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKC, has a negative effect on cell proliferation. However, the mode of PKC, activation, the cell cycle stage requiring PKC, activity, and the exact role of PKC, at that stage remains unknown. Results: Here we show that the treatment of quiescent fibroblasts with serum activates PKC, at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKC,. Importantly, an inhibitor of PKC,, rottlerin, suppresses the biphasic activation of PKC,, and suppression of the second phase of PKC, activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKC, mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKC, plays a positive role in cell cycle progression. While the over-expression of PKC, enhances serum-induced DNA synthesis, this was not observed for PKC,. Similar experiments using a series of PKC,/, chimeras showed that the carboxyl-terminal 51 amino acids of PKC, are responsible for the stimulatory effect. On the other hand, the over-expression of PKC, suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors. Conclusions: We conclude that PKC, plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase. [source] Factor XI deficiency in animal modelsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009T. RENNÉ Summary., The blood coagulation system forms fibrin to limit blood loss from sites of injury, but also contributes to occlusive diseases such as deep vein thrombosis, myocardial infarction, and stroke. In the current model of a coagulation balance, normal hemostasis and thrombosis represent two sides of the same coin; however, data from coagulation factor XI-deficient animal models have challenged this dogma. Gene targeting of factor XI, a serine protease of the intrinsic pathway of coagulation, severely impairs arterial thrombus formation but is not associated with excessive bleeding. Mechanistically, factor XI may be activated by factor XII following contact activation or by thrombin in a feedback activation loop. This review focuses on the role of factor XI, and its deficiency states as novel target for prevention of thrombosis with low bleeding risk in animal models. [source] An electrostatic network and long-range regulation of Src kinasesPROTEIN SCIENCE, Issue 11 2008Elif Ozkirimli Abstract The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain,domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. [source] A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulationPROTEIN SCIENCE, Issue 4 2007Ole H. Olsen Abstract Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365,374{216,225}) of the so-called activation domain and the 170-loop (residues 313,322{170A,175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S1 and S3 substrate pockets, the activation pocket, and N-terminal insertion. [source] Src kinase activation: A switched electrostatic networkPROTEIN SCIENCE, Issue 5 2006Elif Ozkirimli Abstract Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an ,-helix (,C), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with ,C. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed. [source] How does activation loop phosphorylation modulate catalytic activity in the cAMP-dependent protein kinase: A theoretical studyPROTEIN SCIENCE, Issue 4 2006Yuhui Cheng Abstract Phosphorylation mediates the function of many proteins and enzymes. In the catalytic subunit of cAMP-dependent protein kinase, phosphorylation of Thr 197 in the activation loop strongly influences its catalytic activity. In order to provide theoretical understanding about this important regulatory process, classical molecular dynamics simulations and ab initio QM/MM calculations have been carried out on the wild-type PKA,Mg2 ATP,substrate complex and its dephosphorylated mutant, T197A. It was found that pThr 197 not only facilitates the phosphoryl transfer reaction by stabilizing the transition state through electrostatic interactions but also strongly affects its essential protein dynamics as well as the active site conformation. [source] The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: Mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175cPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2008Marc J. Canova Abstract Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis. [source] Regulation of the epididymal receptor tyrosine kinase ros by the protein tyrosine phosphatase SHP-1AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2002Rico Pusch The SH2 domain PTP SHP-1 was recently identified as a potent negative regulator of the orphan receptor tyrosine kinase Ros, an important regulator of epidimys differentiation (Keilhack et al. J Cell Biol 2001; 152:325,334). Phosphorylated Ros strongly and directly associates with SHP-1 in yeast-two-hybrid, GST pull-down, and coimmunoprecipitation experiments. Catalytically inactive SHP-1C455S exhibits greatly elevated binding to phosphorylated Ros. Direct Ros,SHP-1 interaction is mediated by the SHP-1 N-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively down-regulates Ros-dependent proliferation and transformation. Elevated phosphorylation of Ros in ,viable motheaten (me-v)' mice which, have strongly reduced SHP-1 activity, suggests that Ros signaling is under control of SHP-1 in vivo. Thus sterility of male me-v mice seems to be related to dysregulation of Ros. A synthetic phosphopeptide derived from the Ros sequence around Y2267 potently activates recombinant SHP-1 in vitro but is not a good substrate for SHP-1. In contrast, phosphorylation sites in the activation loop of Ros are effectively dephosphorylated. Based on these observations we propose a mechanistic model of Ros,SHP-1 interaction. Using fusion proteins of SHP-1 variants and of Ros with GFP-proteins of different spectral characteristics the interaction of Ros and SHP-1 can be visualized in intact cells by different microscopic techniques. [source] Structures of human MST3 kinase in complex with adenine, ADP and Mn2+ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010Tzu-Ping Ko The MST family is a subclass of mammalian serine/threonine kinases that are related to the yeast sterile-20 protein and are implicated in regulating cell growth and transformation. The MST3 protein contains a 300-residue catalytic domain and a 130-residue regulatory domain, which can be cleaved by caspase and activated by autophosphorylation, promoting apoptosis. Here, five crystal structures of the catalytic domain of MST3 are presented, including a complex with ADP and manganese, a unique cofactor preferred by the enzyme, and a complex with adenine. Similar to other protein kinases, the catalytic domain of MST3 folds into two lobes: the smaller N lobe forms the nucleotide-binding site and the larger C lobe recognizes the polypeptide substrate. The bound ADP and Mn2+ ions are covered by a glycine-rich loop and held in place by Asn149 and Asp162. A different orientation was observed for the ligand in the MST3,adenine complex. In the activation loop, the side chain of Thr178 is phosphorylated and is sandwiched by Arg143 and Arg176. Comparison of this structure with other similar kinase structures shows a 180° rotation of the loop, leading to activation of the enzyme. The well defined protein,ligand interactions also provide useful information for the design of potent inhibitors. [source] Structural basis for the high-affinity binding of pyrrolotriazine inhibitors of p38 MAP kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2008John S. Sack The crystal structure of unphosphorylated p38, MAP kinase complexed with a representative pyrrolotriazine-based inhibitor led to the elucidation of the high-affinity binding mode of this class of compounds at the ATP-binding site. The ligand binds in an extended conformation, with one end interacting with the adenine-pocket hinge region, including a hydrogen bond from the carboxyl O atom of Met109. The other end of the ligand interacts with the hydrophobic pocket of the binding site and with the backbone N atom of Asp168 in the DFG activation loop. Addition of an extended benzylmorpholine group forces the DFG loop to flip out of position and allows the ligand to make additional interactions with the protein. [source] A conserved mechanism of autoinhibition for the AMPK kinase domain: ATP-binding site and catalytic loop refolding as a means of regulationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Dene R. Littler The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9,Å resolution crystal structure of the isolated kinase domain from the ,2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPK,2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function. [source] Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of a Chlamydia trachomatis OmpR/PhoB-subfamily response regulator homolog, ChxRACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009John M. Hickey Two-component signal transduction systems in bacteria are a primary mechanism for responding to environmental stimuli and adjusting gene expression accordingly. Generally in these systems a sensor kinase phosphorylates a response regulator that regulates transcription. Response regulators contain two domains: a receiver domain and an effector domain. The receiver domain is typically phosphorylated and as a result facilitates the DNA-binding and transcriptional activity of the effector domain. The OmpR/PhoB subfamily is the largest of the response-regulator subfamilies and is primarily defined by the winged helix,turn,helix DNA-binding motif within the effector domain. The overall structure of effector domains is highly conserved and contains three defined elements that are critical for transcriptional regulation: a DNA major-groove binding helix, a DNA minor-groove binding wing and a transcriptional activation loop. These functional elements are often diverse in sequence and conformation and reflect the functional differences observed between individual subfamily members. ChxR from Chlamydia trachomatis is an atypical OmpR/PhoB response regulator homolog that has transcriptional activity in the absence of phosphorylation. To facilitate the precise identification of the functional elements of the ChxR effector domain, this protein was cloned, expressed, purified and crystallized. Crystals were obtained from two separate mother liquors, producing two morphologically distinct crystals. The space group of both crystals was P43212 (or its enantiomorph P41212) with isomorphous unit-cell parameters; the crystals diffracted to 2.2,2.5,Å resolution. [source] Mechanisms of constitutive activation of Janus kinase 2-V617F revealed at the atomic level through molecular dynamics simulationsCANCER, Issue 8 2009Tai-Sung Lee PhD Abstract BACKGROUND: The tyrosine kinase Janus kinase 2 (JAK2) is important in triggering nuclear translocation and regulation of target genes expression through signal transducer and activator of transcription pathways. The valine-to-phenylalanine mutation at amino acid 617 (V617F), which results in the deregulation of JAK2, has been implicated in the oncogenesis of chronic myeloproliferative disease. However, both the mechanism of JAK2 autoinhibition and the mechanism of V617F constitutive activation remain unclear. METHOD: In this work, the authors used molecular dynamics simulation techniques to establish plausible mechanisms of JAK2 autoinhibition and V617F constitutive activation at the atomic level. RESULTS: In wild-type JAK2, the activation loop of JAK2-homology domain 1 (JH1) is pulled toward the JH1/JH2 interface through interactions with key residues of JH2, especially S591, F595, and V617, and stabilizes the inactivated form of JH1. In the case of V617F, through the aromatic ring-ring stacking interaction, F617 blocks the interaction of JH1 the activation loop, S591, and F595, thus causing the JH1 activation loop to move back to its activated form. CONCLUSIONS: The current results indicated that this simulation-derived mechanism of JAK2 autoregulation is consistent with current available experimental evidence and may lead to a deeper understanding of JAK2 and other kinase systems that are regulated by pseudokinases. Cancer 2009. © 2009 American Cancer Society. [source] | |||||||||||||||||||||||||