Growth factor signaling is mediated through Class IA phosphatidylinositol 3-kinases (PI3Ks). Among this class of enzymes, only p110␣, encoded by the PIK3CA gene, has been found to be mutant in human cancers. To determine the specific functions of p110␣, we generated mice carrying a conditionally targeted allele of the PIK3CA gene. Here, we report that PIK3CA-knockout mouse embryonic fibroblasts are deficient in cellular signaling in response to various growth factors, unable to differentiate into adipocytes, and resistant to oncogenic transformation induced by a variety of oncogenic receptor tyrosine kinases, indicating a fundamental role for p110␣ in these biological processes.adipocyte differentiation ͉ cancer therapy ͉ conditional knockout ͉ PIK3CA C lass IA PI3Ks are heterodimeric lipid kinases consisting of a p110 catalytic subunit complexed to one of a family of regulatory subunits (p85␣, p55␣, p50␣, p85␤, p55␥), collectively called p85 (1, 2). In response to growth factor stimulation and the subsequent activation of receptor tyrosine kinases (RTKs), class IA PI3Ks are recruited to the membrane via interaction of the p85 subunit with phosphotyrosine-containing motifs on the activated receptor. The p110 catalytic subunit of PI3K then catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to form phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). The lipid second messenger PIP 3 in turn activates the Ser͞Thr kinase AKT and other downstream effectors to regulate multiple cellular functions, including proliferation, survival and migration. There are three isoforms of the p110 catalytic subunit (␣, ␤, and ␦) that are encoded by the PIK3CA, PIK3CB, and PIK3CD genes, respectively. Both p110␣ and p110␤ isoforms are ubiquitously expressed, whereas the p110␦ subunit is primarily found in leukocytes (3, 4). Notably, among these isoforms, only p110␣ has been found to be frequently mutated in human tumors (5), suggesting distinct roles for the individual p110 isoforms in both normal signaling and oncogenic transformation. Previous efforts using gene targeting in mice have revealed that p110␦ plays essential roles in the development and function of lymphocytes, mast cells and possibly neutrophils (6-10). However, mice lacking p110␣ or p110␤ die early in embryonic development (11,12), which has precluded delineating the functions of the individual p110␣ and p110␤ isoforms by genetic ablation. Recent articles have used either a mouse heterozygous for the knockin of a kinase dead allele of p110␣ (13) or small molecule inhibitors of PI3K-p110␣ (14) to study its role in insulin signaling. Here, we report, for the first time, the effects of complete genetic ablation of PI3K-p110␣ on signaling elicited by a panel of growth factors, adipocyte differentiation and oncogenic transformation. Strikingly, we observe that knockout of this single isoform is capable of blocking both normal and oncogenic growth factor signal pathways. Results and DiscussionTo investigate the specific role of p110␣ in signaling an...

Sphingosine 1-phosphate (S1P), produced by two sphingosine kinase isoenzymes, denoted SphK1 and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK1, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethylsphingosine, a competitive inhibitor of both SphKs, implicating S1P generation in this process. Down-regulating expression of SphK1 in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.

Insulin-like growth factor-binding protein-3 (IGFBP-3), a major regulator of endocrine actions of IGFs, is a p53-regulated potent apoptotic factor and is significantly suppressed in a variety of cancers. Recent epidemiologic studies suggest that IGFBP-3 contributes to cancer risk protection in a variety of cancers, and a polymorphic variation of IGFBP-3 influences cancer risk, although other studies vary in their conclusions. Some antiproliferative actions of IGFBP-3 have been reported to be independent of IGFs, but the precise biochemical/molecular mechanisms of IGF-independent, antiproliferative actions of IGFBP-3 are largely unknown. Here we report a new cell death receptor, IGFBP-3R, that is a singlespan membrane protein and binds specifically to IGFBP-3 but not other IGFBP species. Expression analysis of IGFBP-3 and IGFBP-3R indicates that the IGFBP-3/IGFBP-3R axis is impaired in breast and prostate cancer. We also provide evidence for anti-tumor effect of IGFBP-3R in vivo using prostate and breast cancer xenografts in athymic nude mice. Further in vitro studies demonstrate that IGFBP-3R mediates IGFBP-3-induced caspase-8-dependent apoptosis in various cancer cells. Knockdown of IGFBP-3R attenuated IGFBP-3-induced caspase activities and apoptosis, whereas overexpression of IGFBP-3R enhanced IGFBP-3 biological effects. IGFBP-3R physically interacts and activates caspase-8, and knockdown of caspase-8 expression or activity inhibited IGFBP-3/IGFBP-3R-induced apoptosis. Here, we propose that IGFBP-3R represents a novel cell death receptor and is essential for the IGFBP-3-induced apoptosis and tumor suppression. Thus, the IGFBP-3/IGFBP-3R axis may provide therapeutic and prognostic value for the treatment of cancer.Insulin-like growth factor-binding proteins (IGFBPs) 3 are integral components of the IGF system and modulate biological actions of IGFs such as cellular proliferation, differentiation, increase in metabolic activity, and cell survival (1). Apart from its ability to inhibit or enhance IGF actions, all the IGFBPs, IGFBP-1 to -6, have been reported to exert distinct biological actions such as cell proliferation, differentiation, migration, angiogenesis, and apoptosis through an IGF/IGF-I receptor (IGF-IR)-independent manner (2-7). These intrinsic biological activities of IGFBPs appear to be critical to cardiogenesis, vascular development, and pathogenesis of cancer.IGFBP-3, the most abundant IGFBP species in serum, circulates as a 150-kDa ternary complex with an acid-labile subunit and IGF peptide (1-3). Classically, the principal function of IGFBP-3 has been to transport IGFs, protecting them from rapid clearance and/or degradation, and modulating IGF bioavailability to cell-surface IGF receptors (8 -9). Many studies have reported that IGFBP-3 exhibits distinct biological effects independent of the IGF/IGF-IR axis, in particular cell growth inhibition and induction of apoptosis in a variety of cancer cells (10 -14). IGFBP-3 was shown to exert its IGF/IGF-IR-independent, antiproliferative actions via ...

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF-or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate G␣ i suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.The bioactive sphingolipid, sphingosine-1-phosphate (S1P), 1 is a ligand for a family of five specific G protein-coupled receptors (S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) (1) that are coupled to various G proteins. These receptors regulate diverse cellular processes and have been implicated in cytoskeletal rearrangement and regulation of cell movement (2, 3). The founding member of this receptor family, S1P 1 , couples to a G i pathway (4), and its gene disruption in mice demonstrated an essential function for vascular maturation (5). More recently, several studies have established that S1P 1 is also essential for lymphocyte recirculation and that it regulates egress from peripheral lymphoid organs (6, 7). In addition to its extracellular functions, S1P acts intracellularly to regulate cell survival (8), yet its direct targets have not yet been elucidated.As with other potent lipid mediators, levels of S1P are low and tightly regulated in a spatial-temporal manner. Sphingosine kinase (SphK) catalyzes the synthesis of S1P (2), which can be irreversibly degraded by S1P lyase (9 -11) and reversibly conver...

Signal transduction by the erythropoietin receptor (EPOR) is activated by ligand-mediated receptor homodimerization. However, the relationship between extracellular and intracellular domain oligomerization remains poorly understood. To assess the requirements for dimerization of receptor cytoplasmic sequences for signaling, we overexpressed mutant EPORs in combination with wild-type (WT) EPOR to drive formation of heterodimeric (i.e. WT-mutant) receptor complexes. Dimerization of the membrane-proximal portion of the EPOR cytoplasmic region was found to be critical for the initiation of mitogenic signaling. However, dimerization of the entire EPOR cytoplasmic region was not required. To examine this process more closely, we generated chimeras between the intracellular and transmembrane portions of the EPOR and the extracellular domains of the interleukin-2 receptor ␤ and ␥ c chains. These chimeras allowed us to assess more precisely the signaling role of each receptor chain because only heterodimers of WT and mutant receptor chimeras form in the presence of interleukin-2. Coexpression studies demonstrated that a functional receptor complex requires the membrane-proximal region of each receptor subunit in the oligomer to permit activation of JAK2 but only one membrane-distal tail to activate STAT5 and to support cell proliferation. Thus, this study defines key relationships involved in the assembly and activation of the EPOR signal transduction complex which may be applicable to other homodimeric cytokine receptors. Erythropoietin (EPO)1 is essential for the survival, proliferation, and terminal differentiation of erythroid progenitor cells (1-3). The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily and as such contains the structural motifs of four cysteine residues with canonical spacing and the sequence WSXWS in the extracellular domain (4, 5). Although protein tyrosine phosphorylation is required for cytokine signaling (6), the cytoplasmic regions of these receptors lack intrinsic enzymatic activity. Signal transduction instead is mediated by cytoplasmic protein tyrosine kinases of the JAK family (7). These kinases are constitutively associated with receptor chains; residues in the membrane-proximal region of the EPOR cytoplasmic tail are required for this association. Ligand binding results in homodimerization of EPOR chains, which is critical for the initiation of intracellular signaling (8 -12).The role of homodimerization in the initiation of signaling is highlighted by constitutively active variants of the EPOR chain (10, 13). In the three mutants described to date, R129C, E132C, and E133C, amino acids in the extracellular domain are replaced by cysteines, leading to the formation of intermolecular disulfide bridges and constitutive signaling. In contrast, a number of other cytokine receptors signal through the heterodimerization of asymmetric receptor chains. For example, interleukin-2 (IL-2) binds and heterodimerizes the IL-2R␤ and ␥ c receptor chains (for review, see Refs. 14...