RESUMEN

Cell adhesion is a multistep process that requires the interaction of integrins with their ligands in cell attachment, the activation of integrin-triggered signals, and cell spreading. Integrin beta subunit cytoplasmic domains (beta tails) participate in regulating each of these steps; however, it is not known whether the same or different regions within beta tails are required. We generated a panel of amino acid substitutions within the beta1 and beta3 cytoplasmic domains to determine whether distinct regions within beta3 tails regulate different steps in adhesion. We expressed these beta cytoplasmic domains in the context of interleukin 2 (IL-2) receptor (tac) chimeras and tested their ability to activate tyrosine phosphorylation, to regulate beta1 integrin conformation and to inhibit beta1 integrin function in cell attachment and spreading. We found that many of the mutant beta3 and beta3 chimeras either had no effect on these parameters or dramatically inhibited the function of the beta tail in most assays. However, one set of analogous Ala substitutions in the beta1 and beta3 tails differentially affected the ability of the tac-beta3 and tac-beta3 chimeras to activate tyrosine phosphorylation. The tac-beta1 mutant containing Ala substitutions for the VTT motif did not signal, whereas the analogous tac-beta3 mutant was able to activate tyrosine phosphorylation, albeit not to wild-type levels. We also identified a few mutations that inhibited beta tail function in only a subset of assays. Ala substitutions for the Val residue in the VTT motif of the beta1 tail or for the conserved Asp and Glu residues in the membrane-proximal region of the beta3 tail greatly diminished the ability of tac-beta1 and tac-beta3 to inhibit cell spreading, but had minimal effects in other assays. Ala substitutions for the Trp and Asp residues in the conserved WDT motif in the beta1 tail had dramatic effects on the ability of tac-beta1 to regulate integrin conformation and function in cell spreading, but had no or intermediate effects in other assays. The identification of mutations in the beta1 and beta3 tails that specifically abrogated the ability of these beta tails to regulate beta1 integrin conformation and function in cell spreading suggests that distinct protein interactions with beta tails regulate beta cytoplasmic domain function in these processes.

Asunto(s)

Fibroblastos/citología , Integrina beta1/química , Integrina beta1/genética , Tirosina/metabolismo , Secuencia de Aminoácidos , Antígenos CD/química , Antígenos CD/genética , Adhesión Celular/fisiología , Tamaño de la Célula/fisiología , Secuencia Conservada , Citoplasma/química , Fibroblastos/metabolismo , Humanos , Integrina beta3 , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fosforilación , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Piel/citología

RESUMEN

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Integrina beta1/química , Integrina beta1/metabolismo , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismo , Sustitución de Aminoácidos/genética , Animales , Células CHO , Adhesión Celular , Tamaño de la Célula , Colágeno/metabolismo , Cricetinae , Citoplasma/química , Activación Enzimática , Fibroblastos , Fibronectinas/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Genes Dominantes/genética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Estructura Terciaria de Proteína , Subunidades de Proteína , Transducción de Señal , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/genética , Proteínas ras/química , Proteínas ras/genética

RESUMEN

The importance of C/EBP proteins in B cell biology is suggested by the occurrence of functionally important C/EBP binding sites in Ig gene enhancers and promoters, and the knowledge that family member NF-IL-6 is induced in other systems in response to regulators of B cell differentiation. We have studied the expression pattern and activity of C/EBP family transcriptional regulators in B cells at different developmental stages by using B cell lines and normal splenic B cells. Two family members, Ig/EBP and NF-IL-6, seem to be the major regulators of C/EBP site-dependent transcriptional activity in B cells. Negative regulator Ig/EBP is predominantly present in early B cells; activator NF-IL-6 increases in more mature B cells and is induced by LPS activation of splenic B cells. LIP, an N-terminally truncated form of NF-IL-6, was found in most B cell lines tested; LIP can act as a weak transcriptional activator in B cell lines. Partly as a result of the differential amounts of C/EBP family proteins, C/EBP sites do not function as activator sites in early B cells but are activator sites in terminally differentiated B cells.

Asunto(s)

Linfocitos B/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular/fisiología , Línea Celular , Regulación hacia Abajo/inmunología , Femenino , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos , ARN Mensajero/biosíntesis , Bazo/citología , Transfección/genética , Regulación hacia Arriba/inmunología

RESUMEN

Extracellular carbonic anhydrase from the unicellular green alga Chlamydomonas reinhardtii is an oligomeric protein containing subunits of 36 and 4 kDa which are joined by disulfide bonds to form higher molecular mass oligomers. In this study, the effect of dithiothreitol on some properties of the enzyme were examined. Dithiothreitol caused a 40% activation of the catalytic activity of the enzyme at low concentrations (0.1 mM), but an inactivation of about 85% of the catalytic activity at high (50 mM) concentrations. Chemical cross-linking of the enzyme with dimethyl suberimidate revealed the existence of oligomers containing up to three large subunits and at least two small subunits. Cross-linking analysis of dithiothreitol-treated carbonic anhydrase revealed that 0.1 mM dithiothreitol had no effect on the subunit composition of the enzyme, but 10 or 50 mM caused subunit dissociation, including the apparent complete dissociation of the small subunits from the large subunits. There was a characteristic enhancement of dansylamide fluorescence when this fluorescent sulfonamide bound carbonic anhydrase and the fluorescence enhancement was retained following the dithiothreitol-induced dissociation of the enzyme. These results indicate that disulfide bonds are essential for maintenance of the oligomeric structure of Chlamydomonas reinhardtii carbonic anhydrase, and that the small subunit may be necessary for enhancing catalysis, but not for the binding of sulfonamides to the enzyme.

Asunto(s)

Anhidrasas Carbónicas/efectos de los fármacos , Chlamydomonas/enzimología , Ditiotreitol/farmacología , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Catálisis/efectos de los fármacos , Reactivos de Enlaces Cruzados , Compuestos de Dansilo , Dimetil Suberimidato , Disulfuros/análisis , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Sustancias Macromoleculares , Unión Proteica/efectos de los fármacos , Sulfonamidas/metabolismo

RESUMEN

The reliability of (13)C NMR chemical shift correlations in the application of the "tool of increasing electron demand" to stable long-lived carbocationic systems is demonstrated by a comprehensive analysis of 22 stable aryl-substituted carbocationic systems. The observation of slopes of less than unity in such chemical shift correlations for several cationic systems has been attributed to additional charge delocalizing mechanisms present in the system (such as homoallylic, cyclopropyl, and pi conjugations). The onset of nonclassical sigma-delocalization in 2-aryl-2-norbornyl cations with electron withdrawing-substituents previously observed was further verified by using sigma(C+) substituent constants. Difficulties in relating the C(alpha)NMR shifts in different carbocationic systems are also discussed.