Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/ mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P 2 ] and PI(3,4,5)P 3 , as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P 2 . The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P 2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P 2 and PI(3,4,5)P 3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.Cellular adhesion to extracellular matrix proteins is mediated by a diverse class of ␣␤ heterodimeric receptors known as integrins. In addition to participating in cellular adhesion, these cell surface receptors transduce signals within the cell to regulate intracellular events, including cytoskeletal rearrangements and cell spreading, migration, differentiation, survival, and cell growth. These events are associated with activation of multiple intracellular signal transduction pathways, involving protein phosphorylation, alterations in calcium levels, changes in cytoplasmic pH, cytoskeletal rearrangements and inositol phospholipid turnover (for reviews, see references 17, 40, and 76). Although many signaling enzymes are activated following integrin engagement, little is known about the specific roles of these proteins in mediating integrin-induced changes in cell behavior.One enzyme that has been implicated in integrin-induced signal transduction is phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme catalyzes the phosphorylation of inositol lipids at the D-3 position of the inositol ring, resulting in the formation of the 3-phosphorylated phosphoinositides (3-PPIs): phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4-bisph...

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.

The gene encoding dARC1, one of three Drosophila homologs of mammalian activity-regulated cytoskeleton-associated protein (ARC), is upregulated in both seizure and muscular hypercontraction mutants. In this study we generate a null mutant for dArc1 and show that this gene is not involved in synaptic plasticity at the larval neuromuscular junction or in formation or decay of short-term memory of courtship conditioning, but rather is a modifier of stress-induced behavior. dARC1 is expressed in a number of neurosecretory cells and mutants are starvation-resistant, exhibiting an increased time of survival in the absence of food. Starvation resistance is likely due to the fact that dArc1 mutants lack the normal hyperlocomotor response to starvation, which is almost universal in the animal kingdom. dARC1 acts in insulin-producing neurons of the pars intercerebralis to control this behavior, but does not appear to be a general regulator of insulin signaling. This suggests that there are multiple modes of communication between the pars and the ring gland that control starvation-induced behavioral responses.

Oxidized low-density lipoprotein (OxLDL) has been implicated as a proatherogenic factor with a pathological role in the induction of endothelial dysfunction. Endothelial cells bind and uptake OxLDL primarily through the scavenger receptor lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1), which is believed to mediate critical effects of OxLDL in endothelial cells. To examine the biological events following LOX-1 activation by OxLDL, we used cDNA microarray analysis to globally analyze gene expression changes induced by OxLDL treatment of human aortic endothelial cell line (HAECT) cells overexpressing LOX-1. Consistent with reported functions of OxLDL, in control HAECT cells, OxLDL elicited gene changes in the oxidative stress pathway and other signaling pathways related to OxLDL. With OxLDL treatment, LOX-1-dependent gene expression changes associated with inflammation, cell adhesion, and signal transduction were observed. The transcripts of a number of cytokines and chemokines were induced, which included interleukin-8, CXCL2, CXCL3, and colony-stimulating factor-3. The secretion of these cytokines was confirmed by enzyme-linked immunosorbent assay analysis. In addition, our data revealed a novel link between LOX-1 and a number of genes, including Delta/notch-like epidermal growth factor repeat containing, stanniocalcin-1, cAMP response element modulator, and dual specificity phosphatase 1. Promoter analysis on the genes that changed as a result of LOX-1 activation by OxLDL allowed us to identify early growth response 1 and cAMP response element-binding protein as potential novel transcription factors that function downstream of LOX-1. Our study has enabled us to elucidate the gene expression changes following OxLDL activation of LOX-1 in endothelial cells and discover novel downstream targets for LOX-1.

Oxidatively modified low-density lipoprotein (OxLDL) is a contributing factor of endothelial dysfunction, an early cellular event during atherogenesis. In endothelial cells, OxLDL has been shown to stimulate proinflammatory responses, increase lipid accumulation, and induce the expression of adhesion and extracellular matrix degrading molecules. The primary receptor for OxLDL on endothelial cells has been identified as a member of the scavenger receptor family called lectin-like OxLDL receptor-1 (LOX-1). A number of studies on LOX-1 have implicated its role in multiple cardiovascular diseases including atherosclerosis. To better understand the molecular mechanisms underlying the role of LOX-1 in endothelial cells, we identified interacting proteins in an affinity-purified LOX-1 receptor complex from human aortic endothelial HAECT cells by mass spectrometry. Two molecules involved in Rho signaling pathway, ARHGEF1 and ROCK2, were identified, and their associations with LOX-1 were confirmed in reciprocal immunoprecipitation studies. Particularly, ROCK2 was found to dynamically associate with LOX-1 in the presence of OxLDL. In addition, OxLDL treatment stimulated ROCK2 catalytic activity, and ROCK2 inhibition attenuated NF-kappaB activation and IL-8 production resulting from OxLDL activation of LOX-1. In summary, a functional proteomics approach has enabled us to identify novel LOX-1 interactors that potentially contribute to the cellular and signaling functions of LOX-1.