Regulation of the Forkhead transcription factor AFX by Ral-dependent phosphorylation of threonines 447 and 451.

AFX is a Forkhead transcription factor that induces a G(1) cell cycle arrest via upregulation of the cell cycle inhibitor p27(Kip1). Previously we have shown that protein kinase B (PKB) phosphorylates AFX causing inhibition of AFX by nuclear exclusion. In addition, Ras, through the activation of the RalGEF-Ral pathway, induces phosphorylation of AFX. Here we show that the Ras-Ral pathway provokes phosphorylation of threonines 447 and 451 in the C terminus of AFX. A mutant protein in which both threonines are substituted for alanines (T447A/T451A) still responds to PKB-regulated nuclear-cytoplasmic shuttling, but transcriptional activity and consequent G(1) cell cycle arrest are greatly impaired. Furthermore, inhibition of the Ral signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Kip1), while activation of Ral augments AFX activity. From these results we conclude that Ral-mediated phosphorylation of threonines 447 and 451 is required for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest by the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphorylation plays a role in the regulation of AFX by PKB.

Ras-dependent regulation of c-Jun phosphorylation is mediated by the Ral guanine nucleotide exchange factor-Ral pathway.

The transcription factor c-Jun is critically involved in the regulation of proliferation and differentiation as well as cellular transformation induced by oncogenic Ras. The signal transduction pathways that couple Ras activation to c-Jun phosphorylation are still partially elusive. Here we show that an activated version of the Ras effector Rlf, a guanine nucleotide exchange factor (GEF) of the small GTPase Ral, can induce the phosphorylation of serines 63 and 73 of c-Jun. In addition, we show that growth factor-induced, Ras-mediated phosphorylation of c-Jun is abolished by inhibitory mutants of the RalGEF-Ral pathway. These results suggest that the RalGEF-Ral pathway plays a major role in Ras-dependent c-Jun phosphorylation. Ral-dependent regulation of c-Jun phosphorylation includes JNK, a still elusive JNKK, and possibly Src.

Direct control of the Forkhead transcription factor AFX by protein kinase B.

The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Here we show that protein kinase B phosphorylates AFX, a human orthologue of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus.

The Rap1 GTPase functions as a regulator of morphogenesis in vivo.

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. We show that Ras1-mediated signaling pathways in Drosophila are not influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. Our experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo.

Stimulation of gene induction and cell growth by the Ras effector Rlf.

Rlf is a ubiquitously expressed distinct relative of RalGDS that interacts with active Ras in vitro. We now demonstrate that Rlf, when co-expressed with Ras mutants, associates in vivo with RasV12 and the effector-domain mutant RasV12G37, but not with RasV12E38 or RasV12C40. Rlf exhibits guanine nucleotide exchange activity towards the small GTPase Ral and, importantly, Rlf-induced Ral activation is stimulated by active Ras. In addition, RasV12 and RasV12G37 synergize with Rlf in the transcriptional activation of the c-fos promoter. Rlf, when targeted to the plasma membrane using the Ras farnesyl attachment site (Rlf-CAAX), is constitutively active, inducing both Ral activation and c-fos promoter activity. Rlf-CAAX-induced gene expression is insensitive to dominant negative Ras and the MEK inhibitor PD98059, and involves activation of the serum response element. Furthermore, expression of Rlf-CAAX is sufficient to induce proliferation of NIH 3T3 cells under low-serum conditions. These data demonstrate that Rlf is an effector of Ras which functions as an exchange factor for Ral. Rlf mediates a distinct Ras-induced signalling pathway to gene induction. Finally, a constitutively active form of Rlf can stimulate transcriptional activation and cell growth.