The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster

Abstract

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.

RalA GTPase and MAP4K4 Function through NDR1 Activation in Stress Response and Apoptotic Signaling

“…In Drosophila, the Ral-Exocyst-Msn-JNK cascade regulates developmental-induced apoptosis [26]. In mammals, RalA mediates TNF-α-induced p38 MAPK activation, and oxidative stress-induced FOXO activation, suggesting that RalA may regulate stress responses [26,31].…”

“…MAP4K4 has been defined as a Stress-Activated MAP Kinase (SAPK), driving the activation of MAPK-JNK cascades [22,29,30]. In Drosophila Msn, the fly counterpart of MAP4K4 [28], is part of a Ral-Sec5-Msn-JNK cascade regulating developmentalinduced apoptosis [26]. However, the role of MAP4K4 as signaling partner of RalA and/or RalB has not been defined yet in human cells.…”

“…MAP4K4 is a serine/threonine protein kinase of the GCK-IV family [21,22], and has been reported to support cellular transformation, tumor cell adhesion and invasion [23][24][25]. MAP4K4 is characterized by a unique C-terminal Citron Homology (CNH) domain [21], which is important for homodimerization and protein-protein interaction with Sec5, MEKK1 and beta1-integrin [10,[26][27][28]. MAP4K4 has been defined as a Stress-Activated MAP Kinase (SAPK), driving the activation of MAPK-JNK cascades [22,29,30].…”

Ral inhibits ligand-independent Notch signaling in Drosophila

“…18 Another study using sec5 mutants showed that through the exocyst effector, Ral regulates JNK signaling and p38 MAPK to prevent apoptosis in sensory organ precursor cells. 13 Although there has not yet been a genetic analysis of RalBP1 effector function, Drosophila Ral does bind in vitro to RalBP1 as well to Sec5. 16 We recently discovered a specific role for Drosophila Ral in Notch signaling.…”

“…[5][6][7][8] Through the exocyst, Ral regulates many cellular processes including secretion, 4 cytokinesis, 9 cytoskeletal remodeling, 10,11 membrane remodeling, 12 and apoptosis. 13,14 There are two separate Ral genes in vertebrates, RalA and RalB, which produce proteins comparable in amino acid sequence (81% identical and 89% similar) and organization of functional units. 1,15 RalA and RalB have different cellular functions through RalA-specific and RalB-specific RalGEFs.…”