RESUMEN
MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Mapeo Epitopo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Células Tumorales CultivadasRESUMEN
Mammalian members related to Saccharomyces cerevisiae serine/threonine kinase STE20 can be divided into two subfamilies based on their structure and function. The PAK subfamily is characterized by an N-terminal p21-binding domain (also known as CRIB domain), a C-terminal kinase domain, and is regulated by the small GTP-binding proteins Rac1 and Cdc42Hs. The second group is represented by the GCK-like members, which contain an N-terminal catalytic domain and lack the p21-binding domain. Some of them have been demonstrated to induce c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade, while others have been shown to be activated by a subset of stress conditions or apoptotic agents, although little is known about their specific function. Here, we have identified a novel human STE20-related serine/threonine kinase, belonging to the GCK-like subfamily. This kinase does not induce the JNK/SAPK pathway, but, instead, inhibits the basal activity of JNK/SAPK, and diminishes its activation in response to human epidermal growth factor (EGF). Therefore, we designated this molecule JIK for JNK/SAPK-inhibitory kinase. The inhibition of JNK/SAPK signaling pathway by JIK was found to occur between the EGF receptor and the small GTP-binding proteins Rac1 and Cdc42Hs. In contrast, JIK does not activate nor does it inhibit ERK2, ERK6, p38, or ERK5. Furthermore, JIK kinase activity is not modulated by any exogenous stimuli, but, interestingly, it is dramatically decreased upon EGF receptor activation. Thus, JIK might represent the first member of the STE20 kinase family whose activity can be negatively regulated by tyrosine kinase receptors, and whose downstream targets inhibit, rather than enhance, JNK/SAPK activation.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas del Tejido Nervioso , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Secuencia de Aminoácidos , Animales , Anisomicina/farmacología , Secuencia de Bases , Células COS , Cartilla de ADN , ADN Complementario , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas Quinasa Quinasa PAM , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
Eps8, a substrate of receptor tyrosine kinases, is an SH3 domain containing protein that plays an important role in mitogenic signaling. To determine the cellular function of eps8, we used the SH3 domain of eps8 to screen a human fibroblast M426 expression library and identified, a full-length cDNA clone of 3.2 kb. We designated this clone e3B1 for eps8 SH3 domain binding protein 1. Northern analysis revealed that expression of e3B1 mRNA was ubiquitous in human tissues. The e3B1 gene encodes a SH3 domain containing protein. We show that anti-e3B1 antibodies detect three cytosolic protein species of 65, 68 and 72 kDa in cell lysate isolated from asynchronously growing NIH3T3 cells. E3B1 binds to the SH3 domain of eps8 and Abl in vitro. We also demonstrated that e3B1 associates with eps8 in vivo. Phosphatase digestion and phosphoamino acid analysis revealed that p65e3B1 is a phosphoserine containing protein and p72e3B1 and p68e3B1 are hyperserine-phosphorylated form of p65e3B1. We further determined that the p65e3B1 was the most abundant in serum-starved NIH/EGFR cells. Time course studies initiated by the addition of epidermal growth factor (EGF) revealed that the p72e3B1 started to accumulate at 4 h, peaked at 8 h and remained high until 24 h. Finally, we demonstrate that NIH/EGFR fibroblasts overexpressing e3B1 grow more slowly relative to matched controls.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , ADN Complementario/genética , Dominios Homologos src/genética , Células 3T3/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , División Celular/genética , Proteínas del Citoesqueleto , ADN Complementario/aislamiento & purificación , Factor de Crecimiento Epidérmico/farmacología , Humanos , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Fosforilación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificaciónRESUMEN
Several signal transducers bind through their SH2 domains to phosphotyrosine-containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor-transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates.