RESUMO
We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.
Assuntos
Moléculas de Adesão Celular/metabolismo , Citoplasma/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Molécula 1 de Adesão Intercelular , Antígeno-1 Associado à Função Linfocitária/química , Dados de Sequência Molecular , Fosforilação , Relação Estrutura-Atividade , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.
Assuntos
Histonas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Cinética , Fosforilase a/metabolismo , Proteína Fosfatase 1 , Proteína Fosfatase 2 , RatosRESUMO
Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.
Assuntos
Fígado/enzimologia , Fosfoproteínas Fosfatases/isolamento & purificação , Poliaminas , Polímeros/farmacologia , Animais , Núcleo Celular/enzimologia , Cromatina/enzimologia , Ativação Enzimática/efeitos dos fármacos , Histonas/fisiologia , Nucleoproteínas/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Polieletrólitos , Proteína Fosfatase 1 , Proteína Fosfatase 2 , RatosRESUMO
src homology 2 (SH2) domains are modules of about 100 amino acid residues and bind to phosphotyrosine-containing motifs in a sequence-specific manner. They play important roles in intracellular signal transduction and represent potential targets for pharmacological intervention. The protein tyrosine kinase p56lck is a member of the src family and is involved in T-cell activation. The crystal structure of its SH2 domain with an 11-residue peptide showed that the phosphotyrosine and the Ile residue at the pY + 3 position are recognized by the SH2 domain. We present here the crystal structure of the SH2 domain of human p56lck in complex with the short phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Ile (pYEEI peptide) at 1.0 A resolution. The structural analysis at atomic resolution reveals that residue Arg134 (alphaA2), which interacts with the phosphotyrosine side-chain, is present in two conformations in the complex. The structure at 1.8 A resolution of the complex with the phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Gly (pYEEG peptide), which is 11 fold less potent, shows another binding mode for the pY + 3 residue as well as rearrangements of the side-chain of Arg196 (EF3) and one of the water molecules at the base of the pY + 3 pocket. The structure of the complex with the short pYEEI peptide at atomic resolution represents a good starting point for the design and optimization of new inhibitors. Comparative structural analysis of many different inhibitor complexes will be an important component of this drug discovery process.
Assuntos
Fosfotirosina/química , Domínios de Homologia de src , Quinases da Família src/química , Sequência de Aminoácidos , Humanos , Ligação de Hidrogênio , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica , Conformação Proteica , Quinases da Família src/metabolismoRESUMO
The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcgammaRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the zeta chains provide docking space for several key signal transduction molecules such as ZAP-70, p56lck, and Shc. To determine if ZAP-70, p56lck, and Shc bind to particular zeta chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Glutationa Transferase/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Células COS , Filaminas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Ligação Proteica , Proteínas/genética , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.
Assuntos
Histonas/metabolismo , Fosfosserina/metabolismo , Proteína Quinase C/metabolismo , Serina/análogos & derivados , Sequência de Aminoácidos , Animais , Bromosuccinimida/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Fosforilação , Ratos , Especificidade por Substrato , Trombina/metabolismoRESUMO
Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.
Assuntos
Peptídeos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Ligação Competitiva , Cinética , Ligantes , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Dados de Sequência Molecular , Peptídeos/química , Fosfotirosina , Ligação Proteica , Proteínas Tirosina Quinases/química , Análise Espectral/métodos , Tirosina/química , Tirosina/metabolismoRESUMO
Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.
Assuntos
Dipeptídeos/síntese química , Inibidores Enzimáticos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosfotirosina/química , Domínios de Homologia de src , Ligação Competitiva , Dipeptídeos/química , Dipeptídeos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.
Assuntos
Dipeptídeos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Domínios de Homologia de src , Cristalografia por Raios X , Dipeptídeos/química , Ligantes , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.
Assuntos
Permeabilidade da Membrana Celular , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fenilalanina/síntese química , Piridonas/síntese química , Domínios de Homologia de src , Células CACO-2 , Cálcio/metabolismo , Humanos , Células Jurkat , Ligantes , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Piridonas/química , Piridonas/farmacologiaRESUMO
Two new questionnaires were devised to investigate dimensions of complaint about tinnitus. Following a factor analysis of data provided by a sample of tinnitus patients who were administered the first questionnaire, the second questionnaire was developed. This included questions concerning coping attitudes and beliefs about tinnitus. The results of the two analyses were similar and they indicated the presence of three main dimensions of complaint (emotional distress, auditory perceptual difficulties, and sleep disturbance). However several smaller factors suggested that complaint was more complex than originally predicted. The second questionnaire successfully discriminated complaining from non-complaining patients.
Assuntos
Zumbido/psicologia , Adolescente , Adulto , Sintomas Afetivos/etiologia , Idoso , Percepção Auditiva , Cognição , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Sono-Vigília/etiologia , Zumbido/complicaçõesRESUMO
Questions are raised about the technical and psychological interpretation of loudness match measures in the assessment of tinnitus "intensity". The effect of hearing threshold on loudness matches expressed in sensation level (SL) was investigated by selecting subjects with different degrees of hearing loss. The loudness match expressed in SL was found to be a function of threshold. Correlations were then determined between psychological scales of tinnitus complaint (reported loudness, distress, intrusiveness, and others) and loudness match expressed in HL, SL, sones, or personal loudness units (PLUs). Only matches expressed in PLUs were significantly correlated with reported loudness or other psychological scales. The PLU transformation, derived from an individually determined loudness function, produces values that are generally independent of other audiometric measures. It is therefore recommended for assessing tinnitus "intensity".
Assuntos
Percepção Sonora , Zumbido/diagnóstico , Adolescente , Adulto , Idoso , Audiometria de Tons Puros , Limiar Auditivo , Transtornos da Audição/diagnóstico , Transtornos da Audição/etiologia , Humanos , Hiperacusia , Pessoa de Meia-Idade , Zumbido/complicações , Zumbido/psicologiaRESUMO
This article investigates the possible role of metaphorical thinking in psychotic delusions. Twenty-five participants with delusions were asked to give an account of how their ideas had formed and to describe recent experiences relevant to their delusional beliefs. The data suggest that for some participants there may have been a crucial period when the person has unusual experiences, psychosocial difficulties, and made attempts involving metaphor/metonymy to understand these experiences. Furthermore, some participants reported very recent unusual experiences using metaphorical terms, and we speculate on the possibility that the content of the metaphors contributes to a continuation of psychotic experience. The data form a series of case illustrations and are exploratory. No generalizations can be made, but the presence of significant metaphors and metonymy in 11 out of 25 case histories suggests the process may be an important one. We end by outlining a theoretical model of how metaphors might contribute to the formation of delusions: it is suggested that delusional statements are intended to be literal statements, but report on experiences transformed by metaphorical meaning. This transformation involves the 'fusion' of conceptual domains.
Assuntos
Transtorno Depressivo/psicologia , Metáfora , Transtornos Psicóticos/psicologia , Esquizofrenia Paranoide/psicologia , Adulto , Idoso , Formação de Conceito , Transtorno Depressivo/diagnóstico , Feminino , Alucinações , Humanos , Entrevista Psicológica , Acontecimentos que Mudam a Vida , Masculino , Pessoa de Meia-Idade , Modelos Psicológicos , Psicologia , Transtornos Psicóticos/diagnóstico , Esquizofrenia Paranoide/diagnóstico , PensamentoRESUMO
The present report is of the relationships between personality measures, hallucinatory experiences, and hypnogogic/hypnopompic imagery in the normal population. 'Psychoticism' was significantly related to reports of hallucinatory experiences, and a measure of hallucinatory predisposition was significantly associated with strong hypnogogic/hypnopompic imagery.
Assuntos
Alucinações/psicologia , Imaginação , Testes de Personalidade , Adulto , Humanos , PsicometriaAssuntos
Terapia de Relaxamento , Zumbido/terapia , Aconselhamento , Depressão/terapia , Humanos , Imaginação , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Previous studies have suggested that cognitive therapy is effective in modifying delusions. AIMS: To assess the effectiveness of cognitive therapy on patients seen in routine clinical work. METHOD: Eighteen patients with chronic delusions were treated using cognitive therapy, after the method of Chadwick and Lowe. A single-case multiple-baseline experimental design was used, including a control treatment. Each subject was used as their own control. RESULTS: Six patients reduced conviction in their delusions during cognitive therapy and not during the control treatment. Seven patients' conviction ratings did not change. Five patients showed a variable response. Degree of conviction did not fall to zero in any patient. All patients reported that the therapy had been helpful; six spontaneously mentioned changes in psychotic thinking. CONCLUSIONS: One-third of patients with chronic delusions whom we treated responded to delusion modification with a reduction in degree of belief. Change within therapy sessions predicted outcome, as did variation in the conviction during baseline. Cognitive therapy with delusions should aim at reducing distress as well as conviction.
Assuntos
Terapia Cognitivo-Comportamental/métodos , Delusões/terapia , Estudos de Casos e Controles , Doença Crônica , Retroalimentação , Humanos , Participação do Paciente , Esquizofrenia Paranoide/terapia , Resultado do TratamentoRESUMO
Multivariate statistical techniques were used to re-analyse the data from the recent DHSS multi-centre masker study. These analyses were undertaken to three ends. First, to clarify and attempt to replicate the previously found factor structure of complaints about tinnitus. Secondly, to attempt to identify common factors in the change or improvement measures pre- and post-masker treatment. Thirdly, to identify predictors of any such outcome factors. Two complaint factors were identified; 'Distress' and 'intrusiveness'. A series of analyses were conducted on change measures using different numbers of subjects and variables. When only semantic differential scales were used, the change factors were very similar to the complaint factors noted above. When variables measuring other aspects of improvement were included, several other factors were identified. These included; 'tinnitus helped', 'masking effects', 'residual inhibition' and 'matched loudness'. Twenty-five conceptually distinct predictors of outcome were identified. These predictor variables were quite different for different outcome factors. For example, high-frequency hearing loss was a predictor of tinnitus being helped by the masker, and a low frequency match and a low masking threshold predicted therapeutic success on residual inhibition. Decrease in matched loudness was predicted by louder tinnitus initially.
Assuntos
Mascaramento Perceptivo , Zumbido/psicologia , Adaptação Psicológica , Aconselhamento , Seguimentos , Auxiliares de Audição , Humanos , Estilo de Vida , Percepção Sonora , Diferencial Semântico , Papel do Doente , Zumbido/terapiaRESUMO
OBJECTIVES: This pilot study describes a qualitative method for exploring delusions in terms of motivational themes. DESIGN: A semi-structured interview schedule was developed on the basis of an elementary conceptual frame specifying research questions. The analysis of each case uses a structured format. Triangulation was used to check: (i) reliability of motive categories; (ii) their consistent application to delusions. METHODS OF ANALYSIS: All patients had delusions and were diagnosed as having a psychotic disorder. Two types of analysis were used: (i) Interpretative phenomenological analysis with features of grounded analysis was used to classify motives. Data from 14 participants was used for this. (ii) The second phase was an examination of a possible correspondence of themes and involved: (a) a category-led thematic analysis of the delusion in terms of motivations; (b) a category-led thematic analysis of life goals and problems again in terms of motivations; and (c) an examination of correspondence between (a) and (b). RESULTS: The classification of goals and difficulties suggested six main categories: social connection; competence; experiential base (i.e. states of mind and body); material base (e.g. housing); direction; and evaluation (i.e. how a person evaluates himself or believes others evaluate him). Four cases are presented, each exploring the correspondence of themes. CONCLUSION: The methods of analysis seemed coherent and useful. In the cases presented, the delusions appeared to relate to fundamental concerns in a person's life.