Luis del Peso

Nod1, an Apaf-1-like Activator of Caspase-9 and Nuclear Factor-κB

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-κB) and bound RICK, a CARD-containing kinase that also induces NF-κB activation. Nod1 mutants inhibited NF-κB activity induced by RICK, but not that resulting from tumor necrosis factor-α stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-κB activation pathways.

RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis.

Signaling through the CD95/Fas/APO-1 death receptor plays a critical role in the homeostasis of the immune system. RICK, a novel protein kinase that regulates CD95-mediated apoptosis was identified and characterized. RICK is composed of an N-terminal serine-threonine kinase catalytic domain and a C-terminal region containing a caspase-recruitment domain. RICK physically interacts with CLARP, a caspase-like molecule known to bind to Fas-associated protein with death domain (FADD) and caspase-8. Expression of RICK promoted the activation of caspase-8 and potentiated apoptosis induced by Fas ligand, FADD, CLARP, and caspase-8. Deletion mutant analysis revealed that both the kinase domain and caspase-recruitment domain were required for RICK to promote apoptosis. Significantly, expression of a RICK mutant in which the lysine of the putative ATP-binding site at position 38 was replaced by a methionine functioned as an inhibitor of CD95-mediated apoptosis. Thus, RICK represents a novel kinase that may regulate apoptosis induced by the CD95/Fas receptor pathway.

Regulation of the forkhead transcription factor FKHR, but not the PAX3-FKHR fusion protein, by the serine/threonine kinase Akt

Akt, a proto-oncogene that encodes a cytosolic serine/threonine kinase, can phosphorylate and modulate the activity of several proteins involved in cellular metabolism and survival. Recently, two mammalian highly related forkhead transcription factors FKHRL1 and AFX and their nematode homologue Daf-16 have been found to be targets of this kinase. Here we show that Akt, but not inactive Akt, represses the transcriptional activity of FKHR, another member of the forkhead family. FKHR mutants with alanine substitutions at three Akt phosphorylation consensus sites (T24, S256 and S319) were inhibited by Akt, but mutation of all three sites rendered FKHR resistant to suppression. By contrast, the transcriptional activity of the oncogenic PAX3 – FKHR fusion protein, containing two consensus phosphorylation sites, was not inhibited by Akt. Importantly, Akt inhibited the translocation of FKHR to the nucleus, providing a mechanism by which Akt might regulate the transcriptional activity of FKHR. Consistent with this model, the localization of the PAX3 – FKHR fusion protein was nuclear and was not altered by Akt. These results provide evidence that Akt inhibits the transcriptional activity of FKHR by controlling its trafficking into the nucleus and that oncogenic PAX3 – FKHR can escape this negative regulation by Akt.

Ras protein is involved in the physiological regulation of phospholipase D by platelet derived growth factor.

Lipid-derived metabolites play an important role in the regulation of cell responses to external stimuli, including cell growth control, transformation and apoptosis. Phospholipase D (PLD) is one of the critical elements in the regulation of lipid metabolism and the generation of second messengers, some of them involved in cell growth control. Oncogenic Ras proteins affect the activity of PLD by two alternate mechanisms, involving a positive activation and a feedback negative loop. Here we investigate the involvement of the proto-oncogenic Ras protein in the physiological activation of PLD induced by platelet-derived growth factor (PDGF). Over-expression of the wild type Ras protein or some of its regulatory components, such as Shc or Grb2, induces an amplification of PLD activation by PDGF challenge. Furthermore, blocking the endogenous Ras by expression of the dominant negative mutant, H-Ras-Asn17 completely eliminated the activation of PLD by PDGF. Thus, PDGF requires a complex system for PLD regulation implying the existence of at least two positive regulatory pathways, a Ras-dependent and a PKC-dependent mechanism. These results imply that PLD is an important element in signaling by Ras proteins that is altered after ras-induced transformation.

Apoptosis and cancer

Apoptosis, or programmed cell death, is a central mechanism controlling cell number and the deletion of unwanted cells. Recent efforts have lead to the identification of the machinery involved in the control and execution of the apoptotic program Alteration of the normal apoptotic program has been identified as the possible cause of a number of human diseases, including neoplasia. In this review the means by which disruption of the normal apoptotic machinery may lead or contribute to tumor generation and progression will be discussed.ResumenLa apoptosis o muerte celular programada es un mecanismo central que controla el nÚmero de células y la eliminatión de las células no deseadas. Algunos trabajos recientes han conducido a la identificatión de los mecanismos que intervienen en el control y la ejecución del programa apoptósico. Se ha identificado la alteración del programa apoptótico normal como posible causa de diversas enfermedades humanas, incluyendo las neoplasias. En esta revisión, se comentará la forma en que la alteración del dispositivo apoptósico normal puede dar lugar o contribuir a producir la generación y progresión de los tumores.