Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8–E3b1–Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure–function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1–dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac–specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 ؊/؊ fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27-42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin-rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 ؊/؊ fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARaselective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARb agonist and an anti-AP1 retinoid have very limited activity, ATRA-and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARa. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD. Cell Death and Differentiation (2000) 7, 447 ± 460.

The 2-phenylaminopyrimidine derivative STI571 is a selective inhibitor of c-Abl, c-kit, and platelet-derived growth factorreceptor tyrosine kinases and is presently in phase II-III clinical studies. Here, this study reports on a novel pharmacologic activity of the compound, ie, enhancement of the cyto-differentiating, growth-inhibitory, and apoptogenic actions of all-trans-retinoic acid (ATRA). Whereas STI571 is not a cytodifferentiating agent by itself, the compound interacts with ATRA and enhances the myeloid maturation program set in motion by the retinoid in the PML-RAR␣ ؉ acute promyelocytic leukemia NB4 and the PML-RAR␣ ؊ myeloblastic HL60 and U937 cell lines. In addition, STI571 relieves the cytodifferentiation block observed in the ATRA-resistant cell lines, NB4.R1, NB4.306, and NB4.007. In NB4 promyelocytes, a RAR␣ agonist, but not an RXR agonist, can substitute for ATRA and interact with STI571. By contrast, STI571 is unique among c-Abl-specific tyrosine kinase inhibitors in modulating the pharmacologic activity of ATRA. In NB4 cells, enhanced cyto-differentiation results in increased up-regulation of the expression of a number of genes coding for myeloid differentiation markers, including CD11b, CD11c, and some of the components of the nicotinamide adenine dinucleotide phosphate-oxidase enzymatic complex. All this is accompanied by inhibition of c-Abl tyrosine phosphorylation and retardation of the retinoid-dependent degradation of PML-RAR␣ and RAR␣. Stabilization of the 2 retinoic acid receptors is likely to be the result of augmented and accelerated inhibition of the proteasome-dependent proteolytic activity observed on ATRA treatment. IntroductionAll-trans-retinoic acid (ATRA) represents the sole example of clinically useful cytodifferentiating agent. 1 Treatment of patients with acute promyelocytic leukemia (APL) with ATRA alone or in combination with chemotherapy results in high rates of complete clinical remission. 1,2 However, the therapeutic use of this compound is limited by a number of problems, which include serious systemic toxicity 3 and induced ATRA resistance. 4,5 In addition, the use of ATRA is limited to the APL subset of acute myelogenous leukemia, as all the other French-American-British subtypes are generally refractory to the cytodifferentiating action of the retinoid. One way to circumvent some or all of the aforementioned problems is to identify agents capable of enhancing the pharmacologic activity of ATRA. In previous reports, we and others demonstrated that agents such as granulocyte colony-stimulating factor, cellpermeable analogs of cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), and interferons enhance the cytodifferentiating action that ATRA exerts on APL and other myeloid cell lines. [6][7][8][9][10][11] The 2-phenylaminopyrimidine derivative STI571 is a tyrosine kinase inhibitor, acting on a restricted number of target proteins, 12,13 ie, c-Abl and its pathologic derivative BCR-Abl, 12,14,15 c-kit 16,17 , and the platelet-derived growth factor (PDGF) receptor. 17,18 In vi...