Autophosphorylation kinetics of protein kinases.
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ABSTRACT:
SUBMITTER: Wang ZX
PROVIDER: S-EPMC1223023 | biostudies-other | 2002 Dec
REPOSITORIES: biostudies-other
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Project description:Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.
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Project description:Receptor-like protein kinases (RLKs) are a large and important group of plant proteins involved in numerous aspects of development and stress response. Within this family, homo-oligermization of receptors followed by autophosphorylation of the intracellular protein kinase domain appears to be a widespread mechanism to regulate protein kinase activity. In vitro studies of several RLKs have identified autophosphorylation sites involved in regulation of catalytic activity and signaling in vivo. Recent work has established that multiple RLKs are biochemically active when expressed in E. coli and readily autophosphorylate prior to purification or subsequent manipulation. This observation has led us to develop a simplified method for assaying RLK phosphorylation status as an indirect measure of intrinsic autophosphorylation activity. The method involves expressing a recombinant RLK protein kinase domain in E. coli, followed by SDS-PAGE of boiled cell lysate, and sequential staining with the phosphoprotein stain Pro-Q Diamond and a colloidal Coomassie total protein stain. We show this method can be used to measure and quantify in vitro autophosphorylation levels of recombinant wildtype and mutant versions of the Arabidopsis RLK HAESA, as well as to detect transphosphorylation activity of recombinant HAESA against a protein kinase inactive version of itself. Our method has several advantages over traditional protein kinase assays. It does not require protein purification, transfer, blotting, or radioactive reagents. It allows for rapid and quantitative assessment of autophosphorylation levels and should have general utility in the study of any autophosphorylating protein kinase expressed in E. coli.
| S-EPMC3702502 | biostudies-literature
Project description:Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an "autophosphorylation complex." We developed and applied a structural bioinformatics method to identify all such autophosphorylation complexes in x-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which five complexes had not previously been described in the publications describing the crystal structures. These five complexes consist of tyrosine residues in the N-terminal juxtamembrane regions of colony-stimulating factor 1 receptor (CSF1R, Tyr(561)) and ephrin receptor A2 (EPHA2, Tyr(594)), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr(394)) and insulin-like growth factor 1 receptor (IGF1R, Tyr(1166)), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser(142)). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore, we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro(447) to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.
| S-EPMC4766099 | biostudies-literature
Project description:Protein phosphorylation, one of the most important post-translational modifications, regulates almost every cellular process. Although signal transduction by protein phosphorylation is extensively studied in Eukaryotes and Bacteria, the knowledge of this process in archaea is greatly lagging behind, especially for Ser/Thr/Tyr phosphorylation by eukaryotic-like protein kinases (ePKs). So far, only a few studies on archaeal ePKs have been reported, most of which focused on the phosphorylation activities in vitro, but their physiological functions and interacting network are still largely unknown. In this study, we systematically investigated the autophosphorylation and cross-phosphorylation activities of ePKs from Sulfolobus islandicus REY15A using proteins expressed in Escherichia coli or S. islandicus. In vitro kinase assay showed that 7 out of the 11 putative ePKs have autophosphorylation activity. A protein Ser/Thr phosphatase, SiRe_1009, was able to dephosphorylate various autophosphorylated ePKs, confirming that these proteins are Ser/Thr kinases. Two ePKs, SiRe_2030 and SiRe_2056, homologs of typical eukaryotic PKs involved in peptide synthesis in response to various cellular stresses, exhibit highly efficient phosphorylation activities on both themselves and other ePKs. Overexpression of the protein kinases in vivo revealed that elevated level of either SiRe_1531 or SiRe_2056 inhibited the cell growth of S. islandicus cells. Finally, a phosphorylation network of the protein kinases was proposed and their putative physiological roles were discussed.
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Project description:BackgroundHomeodomain interacting protein kinases (HIPKs) function as modulators of cellular stress responses and regulate cell differentiation, proliferation and apoptosis. The HIPK family includes HIPK1, HIPK2 and HIPK3, which share a similar domain structure, and the more distantly related HIPK4. Although HIPKs phosphorylate their substrates on serine or threonine residues, it was recently reported that HIPK2 depends on the autophosphorylation of a conserved tyrosine in the activation loop to acquire full catalytic activity and correct subcellular localization. In this study we addressed the question whether tyrosine autophosphorylation in the activation loop has a similar function in the other members of the HIPK family.ResultsAll HIPKs contained phosphotyrosine when expressed in HeLa cells. Catalytically inactive point mutants were not tyrosine-phosphorylated, indicating that HIPKs are dual-specificity protein kinases that autophosphorylate on tyrosine residues. HIPK point mutants lacking the conserved tyrosine residue in the activation loop showed reduced catalytic activity towards peptide and protein substrates. Analysis of these mutants revealed that HIPK1, HIPK2 and HIPK3 but not HIPK4 are capable of autophosphorylating on other tyrosines. Inhibition of tyrosine phosphatase activity by treatment with vanadate enhanced global phosphotyrosine content of HIPK1, HIPK2 and HIPK3 but did not affect tyrosine phosphorylation in the activation loop. Mutation of the activation-loop tyrosines resulted in a redistribution of HIPK1 and HIPK2 from a speckle-like subnuclear compartment to the cytoplasm, whereas catalytically inactive point mutants showed the same pattern of cellular distribution as the wild type proteins. In contrast, mutation of the activating tyrosine did not increase the low percentage of cells with extranuclear HIPK3. HIPK4 was excluded from the nucleus with no difference between the wild type kinase and the point mutants.ConclusionsThese results show that HIPKs share the mechanism of activation by tyrosine autophosphorylation with the closely related DYRK family (dual-specificity tyrosine phosphorylation regulated kinase). However, members of the HIPK family differ regarding the subcellular localization and its dependence on tyrosine autophosphorylation.
| S-EPMC4353451 | biostudies-literature
Project description:In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10(5)-fold range of rate constants (kphos/KS) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate.
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Project description:Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain.
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Project description:The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2?.
| S-EPMC6615999 | biostudies-literature
Project description:DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.
| S-EPMC4054291 | biostudies-literature
Project description:Protein kinases represent a family of enzymes that are critical in cell signaling. One mechanism by which protein kinases are regulated is via autophosphorylation. In the studies described here, we have examined the mechanism of autophosphosphorylation at serine 338 in the regulation of protein kinase A (PKA). Expressed protein ligation allowed for the covalent linkage of an ATP moiety to a Ser mimic at this phosphorylation site. Using a combination of size exclusion chromatography, fluorescence nucleotide binding, kinase measurements, and limited proteolysis assays on this semisynthetic ATP-linked protein, we have obtained unique evidence for an intramolecular autophosphorylation mechanism in PKA regulation. Computational analysis provided a plausible model for a PKA conformation consistent with intramolecular phosphoryl transfer. This approach could be applied to other autoprocessing enzymes by exploiting appropriate transition state analogue motifs in the context of protein semisynthesis.
| S-EPMC2654778 | biostudies-literature