RESUMO
Additive manufacturing of alloys enables low-volume production of functional metallic components with complex geometries. Ultrasonic testing can ensure the quality of these components and detect typical defects generated during laser powder bed fusion (LPBF). However, it is difficult to find a single ultrasonic inspection technique that can detect defects in the large variety of geometries generated using LPBF. In this work, phased array ultrasonic testing (PAUT) is suggested to inspect thick LPBF components, while guided waves are explored for thin curved ones. PAUT is used to detect cylindrical lack of fusion defects in thick LPBF rectangular parts. Practical defects are generated by reducing the laser power at prespecified locations in the samples. The defects' shape and density are verified using optical microscopy and X-ray computed tomography. Partially fused defects down to 0.25 mm in diameter are experimentally detected using a 10 MHz PAUT probe with the total focusing method post-processing. The experimental results are compared to defect images predicted by finite element simulations. For thin components with curved geometry, guided waves are used to detect powder-filled cylindrical defects. The waves are generated using piezoelectric transducers, and the spatiotemporal wavefield is measured using a scanning laser Doppler vibrometer. Using root-mean-square imaging of the wavefield, defects down to 1 mm are clearly detected despite the complex internal features in the samples.
RESUMO
Various human skull models feature a layered cranial structure composed of homogeneous cortical tables and the inner diploë. However, there is a lack of fundamental validation work of such three-layer cranial bone models by combining high-fidelity computational modeling and rigorous experiments. Here, non-contact vibration experiments are conducted on an assortment of dry bone segments from the largest cranial bone regions (parietal, frontal, occipital, and temporal) to estimate the first handful of modal frequencies and damping ratios, as well as mode shapes, in the audio frequency regime. Numerical models that consider the cortical tables and the diploë as domains with separate isotropic material properties are constructed for each bone segment using a routine that identifies the cortical table-diploë boundaries from micro-computed tomography scan images, and reconstructs a three-dimensional geometry layer by layer. The material properties for cortical tables and diploë are obtained using a Hounsfield Unit-based mass density calculation combined with a parameter identification scheme for Young's modulus estimation. With the identified parameters, the average error between experimental and numerical modal frequencies is 1.3% and the modal assurance criterion values for most modes are above 0.90, indicating that the layered model is suitable for predicting the vibrational behavior of cranial bone. The proposed layered modeling and identified elastic parameters are also useful to support computational modeling of cranial guided waves and mode conversion in medical ultrasound. Additionally, the diploë elastic properties are rarely reported in the literature, making this work a fundamental characterization effort that can guide in the selection of material properties for human head models that consider layered cranial bone.
Assuntos
Crânio , Vibração , Osso Esponjoso , Módulo de Elasticidade , Humanos , Crânio/diagnóstico por imagem , Microtomografia por Raio-XRESUMO
Absorbers suppress reflection and scattering of an incident wave by dissipating its energy into heat. As material absorption goes to zero, the energy impinging on an object is necessarily transmitted or scattered away. Specific forms of temporal modulation of the impinging signal can suppress wave scattering and transmission in the transient regime, mimicking the response of a perfect absorber without relying on material loss. This virtual absorption can store energy with large efficiency in a lossless material and then release it on demand. Here, we extend this concept to elastodynamics and experimentally show that longitudinal motion can be perfectly absorbed using a lossless elastic cavity. This energy is then released symmetrically or asymmetrically by controlling the relative phase of the impinging signals. Our work opens previously unexplored pathways for elastodynamic wave control and energy storage, which may be translated to other phononic and photonic systems of technological relevance.
RESUMO
CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.
Assuntos
Caseína Quinase II/metabolismo , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
CK2 is a multitask kinase whose role is essential for a countless number of cellular processes, many of which are critical for blood cell development. A prevailing task for this kinase rests on counteracting programmed cell death triggered by multiple stimuli. CK2 is overexpressed in many solid tumors and in vivo mouse models have proven its tumorigenic potential. Recent data have suggested that CK2 may also have a significant role in the pathogenesis of hematopoietic tumors, such as multiple myeloma, chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia and chronic myeloproliferative neoplasms. CK2 regulates hematopoiesis-associated signaling pathways and seems to reinforce biochemical cascades indispensable for tumor growth, proliferation and resistance to conventional and novel cytotoxic agents. Although its activity is multifold, recent evidence supports the rationale of CK2 inhibition as a therapeutic strategy in solid and hematological tumors and phase-I clinical trials are in progress to test the efficacy of this innovative therapeutic approach. In this review, we will summarize the data supporting CK2 as an oncogenic kinase in blood tumors and we will describe some critical signaling pathways, whose regulation by this protein kinase may be implicated in tumorigenesis.
Assuntos
Caseína Quinase II/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/patologia , Oncogenes/fisiologia , Transdução de Sinais , Animais , Sobrevivência Celular , Neoplasias Hematológicas/enzimologia , Humanos , CamundongosRESUMO
p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.
Assuntos
Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Catálise , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-akt/genética , Serina/metabolismo , Transdução de Sinais , Transdução GenéticaRESUMO
Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Linfócitos T/patologia , Animais , Antibióticos Antineoplásicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Western Blotting , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/metabolismo , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Fosforilação , RNA Interferente Pequeno/farmacologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Transfecção , Vimblastina/farmacologiaRESUMO
Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima , Sequência de Aminoácidos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Caseína Quinase II/genética , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Indução Enzimática , Humanos , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Interferência de RNA , Transdução de Sinais/efeitos dos fármacosRESUMO
Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Caseína Quinase II , Domínio Catalítico , Proteínas de Choque Térmico HSP70/genética , Humanos , Lisina , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Peptídeos/metabolismo , Subunidades Proteicas , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
CK2 is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits.
Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Tirosina , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Caseína Quinase II , Emodina/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli , Heparina/farmacologia , Cinética , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fenilalanina , Fosforilação , Fosfotirosina/metabolismo , Conformação Proteica , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr- or Tyr-specific. In the presence of 10 microM TBB (and 100 microM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3 beta and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC(50) values one--two orders of magnitude higher than CK2 (IC(50)=0.9 microM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays.
Assuntos
Trifosfato de Adenosina , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Triazóis/farmacologia , Substituição de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Caseína Quinase II , Emodina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Jurkat/citologia , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estaurosporina/farmacologia , Especificidade por Substrato , Triazóis/metabolismoRESUMO
On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.
Assuntos
Príons/química , Proteínas Serina-Treonina Quinases/química , Animais , Western Blotting , Calmodulina/metabolismo , Caseína Quinase II , Domínio Catalítico , Bovinos , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Peptídeos/metabolismo , Fosforilação , Príons/metabolismo , Ligação Proteica , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Temperatura , Fatores de TempoRESUMO
Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 alpha subunit alone (Km = 47 nM) or CK2 holoenzyme, tested in the presence of polylysine (Km = 400 nM). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208-402), as judged from experiments with deleted forms of HS1. Ser/Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Treonina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Plaquetas/metabolismo , Western Blotting , Caseína Quinase II , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Dados de Sequência Molecular , Ácido Okadáico/farmacologia , Fosfatos/farmacologia , Fosforilação/efeitos dos fármacos , Polilisina/metabolismo , Testes de Precipitina , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
To assess the functional role of the four conserved cysteinyl residues in the regulatory beta-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated beta-subunit has lost its ability to form either homodimers or regular alpha(2)beta(2) heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine. The pCMB-treated beta-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the alpha-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme. These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme.
Assuntos
Cisteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ácido p-Cloromercurobenzoico/farmacologia , Animais , Caseína Quinase II , Domínio Catalítico , Citosol/enzimologia , Dimerização , Cinética , Fígado/enzimologia , Substâncias Macromoleculares , Fosforilação , Estrutura Quaternária de Proteína , RatosRESUMO
The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Regulação para Baixo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Apoptose , Western Blotting , Calmodulina/metabolismo , Caseína Quinase II , Escherichia coli/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Peptídeos/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Fatores de Tempo , TransfecçãoRESUMO
Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.
Assuntos
Interleucina-1/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Interleucina-1/metabolismo , Sítios de Ligação , Sequência Consenso , Ativação Enzimática , Humanos , Interleucina-1/metabolismo , NF-kappa B/metabolismo , Osteossarcoma , Fosforilação , Fosfotirosina/metabolismo , Testes de Precipitina , Ligação Proteica , Receptores de Interleucina-1/química , Receptores Tipo I de Interleucina-1 , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Domínios de Homologia de src/fisiologiaRESUMO
The SH2 domain of c-Fgr (class 1A) has been expressed in E. coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.
Assuntos
Proteínas Sanguíneas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/química , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Ligação Competitiva , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Expressão Gênica , Glutationa Transferase/genética , Inositol 1,4,5-Trifosfato/farmacologia , Dados de Sequência Molecular , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Recombinantes de Fusão , Relação Estrutura-Atividade , Quinases da Família src/metabolismoRESUMO
Two tyrosyl residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family: autophosphorylation of Tyr416 (c-Src numbering) located in the catalytic domain correlates with enzyme activation, while Csk-mediated phosphorylation of the C-terminal tyrosine Tyr527 (c-Src numbering) gives rise to inactive forms of Src kinases. Here we show that the Src-related Lyn kinase undergoes spontaneous and stoichiometric autophosphorylation at both Tyr396 (homologous to c-Src Tyr416) and Tyr507 (homologous to c-Src Tyr527). Such a doubly autophosphorylated form of Lyn is hyperactive toward peptide substrates and insensitive to Csk-induced downregulation. In contrast, doubly autophosphorylated Lyn exhibits reduced activity toward protein substrates such as phospho-p50/HS1 (hematopoietic-lineage cell-specific protein) and p57/PDI (protein disulfide isomerase related protein), whose multiple sequential/processive phosphorylation relies on the accessibility of the SH2 domain of the kinase. These data disclose a novel conformation of Lyn that is catalytically active despite the presence of an intramolecular interaction between the phosphorylated tail and the SH2 domain. This enzyme conformation is expected to display a reduced oncogenic potential resulting from its defective recognition of a subset of protein substrates whose targeting is mediated by the Lyn SH2 domain.
Assuntos
Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Tirosina Quinase CSK , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Fosforilação , Proteínas Tirosina Quinases/farmacologia , Ratos , Especificidade por Substrato , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Src-related protein kinases, is phosphorylated by C-terminal Src kinase (CSK) and by the two Src-related protein kinases c-Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C-terminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117-124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild-type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position -1 (instead of glutamine), while lysine at position -3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C-terminal peptides, which exhibit relatively high K(m) values, a polyoma-virus-middle-T-antigen-(mT)-derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low K(m) value (63 microM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c-Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n-1. In sharp contrast, TPKIIB/p38syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n-1 included. These data support the notion that the features determining the specific phosphorylation of the C-terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C-terminal tyrosine residue of Src kinases may exist.
Assuntos
Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Quinases da Família src/química , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Quinases da Família src/metabolismoRESUMO
Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.