RESUMEN
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Ácido Aspártico/genética , Caspasa 3 , Línea Celular Tumoral , Membrana Celular/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Doxorrubicina/farmacología , Activación Enzimática , Expresión Génica , Humanos , Células Jurkat , Ratones , Mutación/genética , Neuronas/citología , Neuronas/enzimología , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras , TransfecciónRESUMEN
Netrin-1 receptors UNC5H (UNC5H1-4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Factores de Crecimiento Nervioso/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Supresoras de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Encéfalo/citología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Caspasas/metabolismo , Catálisis , Células Cultivadas , Proteínas Quinasas Asociadas a Muerte Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Receptores de Netrina , Netrina-1 , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/farmacologíaRESUMEN
The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.