A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth.

Here, we report the identification and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of fibroblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential effector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1.

Activation of acid sphingomyelinase by interleukin-1 (IL-1) requires the IL-1 receptor accessory protein.

The cytokine interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune responses, but the mechanisms of its signal transduction and cell activation processes are incompletely understood. Ceramide generated by sphingomyelinases (SMases) is known to function as an important second messenger molecule in the signaling pathway of IL-1 and tumor necrosis factor. To investigate the activation of SMases by IL-1, we used an IL-1 receptor type I (IL-1RI)-positive EL4 thymoma cell line, which is defective in IL-1R accessory protein (IL-1RAcP) expression. In this cell line (EL4D6/76), tumor necrosis factor induced ligand/receptor internalization, NFkappaB nuclear translocation, IL-2 production, and the activation of neutral (N)-SMase and acid (A)-SMase. In contrast, stimulation with IL-1 resulted only in the activation of N-SMase whereas ligand/receptor internalization, NFkappaB translocation, IL-2 production, and activation of A-SMase were not detected. Transfection of this functionally defective EL4D6/76 with IL-1RAcP cDNA restored these functions. These data suggest that A-SMase activity is strongly linked with the internalization of IL-1RI mediated by IL-1RAcP and that A-SMase and N-SMase are activated by different pathways.

The interleukin-1 receptor accessory protein (IL-1RAcP) is essential for IL-1-induced activation of interleukin-1 receptor-associated kinase (IRAK) and stress-activated protein kinases (SAP kinases).

Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL-1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFkappaB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression.

A critical role for interleukin-1 receptor accessory protein in interleukin-1 signaling.

Interleukin-1 (IL-1) is a central molecule in inflammation and immune responses whose pleiotropic activities are mediated by the type I IL-1 receptor (IL-1RI). The IL-1RI alone on the cell surface is silent after binding of the ligand. We show that the recently identified IL-1RI accessory protein (IL-1RAcP) converts the silent into a fully functional IL-1RI complex. Although transfection of IL-1RAcP into IL-1RAcP-deficient EL4D6/76 cells did not alter the binding kinetics or dissociation constants of the 125I-labeled IL-1alpha/IL-1RI complex, a very early event, internalization of the activated receptor complex, and a late event, IL-1-stimulated IL-2 production, were successfully restored. Therefore, recruitment of IL-1RAcP is a critical early step in the signaling cascade mediated by the IL-1RI activation complex.