Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), a substrate for both enzymes, is converted to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P 3 activates purified PLC-␥ isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-␥ isozymes indirectly, in the absence of PLC-␥ tyrosine phosphorylation, through the generation of PI(3,4,5)P 3 .Activation of both PLC 1 and PI 3-kinase often occurs in response to stimulation of cells by a variety of agonists. PLC catalyzes the hydrolysis of PI(4,5)P 2 to generate the second messengers inositol 1,4,5-trisphosphate (I(1,4,5)P 3 ) and diacylglycerol (1-3). PI 3-kinase phosphorylates the D-3 position of PI(4,5)P 2 to produce PI(3,4,5)P 3 , which is then sequentially dephosphorylated to PI(3,4)P 2 and phosphatidylinositol 3-phosphate (4 -7). The activation of each of these two enzymes has been implicated in such diverse cellular processes as mitogenesis, chemotaxis, secretion, and cytoskeletal assembly (4 -7).The phosphoinositides PI(3,4)P 2 and PI(3,4,5)P 3 are not substrates of any known PLC (8) and are normally absent from resting cells; however, they appear within seconds to minutes of stimulation of cells with various growth factors or other cellular activators. In contrast, the concentration of phosphatidylinositol 3-phosphate does not change substantially in response to cell stimulation (4 -7). It has thus been suggested that PI(3,4)P 2 and PI(3,4,5)P 3 might function as intracellular messengers (4 -7). With regard to potential targets of these D-3-phosphorylated lipids, they have been shown to activate Ca 2ϩ -independent isoforms of protein kinase C (9, 10) as well as to bind the pleckstrin homology (PH) domain of the protein serine-threonine kinase Akt, thereby activating its kinase activity (11-13), and to the SH2 domains of the 85-kDa (p85) subunit of PI 3-kinase, thereby preventing its binding to tyrosine-phosphorylated proteins (14).The 10 mammalian PLC isozymes identified to date are single polypeptides and can be divided into three types: PLC-␤, PLC-␥, and PLC-␦ (1). All contain a PH domain in their NH 2 -terminal region. The ␥ type isozymes differ from the other two types in that they contain two SH2 domains, one SH3 domain, and an additional PH domain that is split by the SH domains; these domains are arranged in the order PH(N)-SH2-SH2-SH3-PH(C), where N and C in parentheses denote NH 2 -and COOHterminal locations, respectively. Upon st...

Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 ␤PIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 ␤PIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 ␤PIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 ␤PIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 ␤PIX, also functions upstream of p85 ␤PIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 ␤PIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 ␤PIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 ␤PIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 ␤PIX complex via the ERK cascade and subsequent translocation of this complex.

Senescent cells have been generally characterized to have improper responsiveness to external stimuli and inefficient uptake of materials compared with presenescent cells, probably by downregulation of receptor-mediated endocytosis. Using transferrin-uptake assay and Western blot of endocytosis-related proteins, we found that a significant decrease of amphiphysin-1 is strongly related to the reduction of receptor-mediated endocytosis in both human diploid fibroblasts of multipassages and hydrogen peroxide-induced premature senescence. We reconfirmed the pivotal role of amphiphysin-1 in senescence by showing that transfection of dominant negative amphiphysin-1 reduces transferrin uptake in presenescent cells and that microinjection of wildtype amphiphysin-1 into senescent cells restores the ability of the transferrin uptake nearly up to that of the presenescent cell. We conclude that the loss of endocytotic activity of senescent cells is directly related to the down-regulation of amphiphysin-1, and its functional restorability can be induced by simple microinjection of amphiphysin-1 gene in the senescent cells.Key words: amphiphysin-1 • endocytosis • senescence • human diploid fibroblast ormal human diploid fibroblasts (HDFs) do not divide indefinitely and eventually lead to an arrest of cell division by a process termed cellular, or replicative, senescence (1, 2). Irreversible growth arrest of senescent cells is suggested to be strongly related to the attenuated response to growth factors (3,4). However, in the case of the epidermal growth factor (EGF), the kinetics for binding in senescent cells still remains active as in early-passaged presenescent cells (5,6). Recently, we reported that up-regulation of caveolin in the senescent cells is partly responsible for the attenuated response to growth factors through the regulation of the downstream signal pathway (7). However, for the receptor-mediated endocytosis, the clathrin-dependent endocytosis system plays the more active and dominant role over the caveolae system. Therefore, it would be worthwhile to monitor the efficiency of clathrindependent receptor-mediated endocytosis in the senescent cells in order to elucidate the exact mode of the attenuated response to growth factors in the senescent cells. N The functional capacity of clathrin-dependent receptor-mediated endocytosis in the senescent HDF can be illustrated by the transferrin uptake model system (8). To reveal the molecular mechanism for the altered endocytosis of the senescent cells, the expression of proteins related to the clathrin-mediated endocytosis was analyzed and the functional recovery of the receptormediated endocytosis in the senescent cells was attempted. MATERIALS AND METHODS AntibodiesMonoclonal antibodies against clathrin heavy chain (C68220), dynamin (D25520), α-adaptin (A43920), β-adaptin (A35620), and transferrin receptor (CD71: SC-7088) were purchased from Cell culture and induction of senescenceHuman foreskin fibroblasts were isolated from a 7-year-old Korean male (10) in our labor...

FGF-2 is involved in cell survival

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