Organization of the actin cytoskeleton in eucaryotic cells is controlled by small GTPases of the Rho family. Rac becomes activated by growth factor stimulation and integrin-mediated cell adhesion to extracellular matrix and is known to have a crucial role in lamellipodia formation, cell spreading and migration. At present, the intracellular pathways that connect cell surface receptors to Rac activation are poorly characterized. It has been reported previously that integrin-mediated cell attachment induces activation of the EGF receptor (EGFR) in the absence of EGF. We demonstrate here that this activation is instrumental for integrin-dependent Rac activation. Thus, we found that cells in which EGFR activity had been inhibited failed to spread and form lamellipodia on fibronectin. Failure to spread coincided with inhibition of adhesion-induced GTP loading of Rac and also with inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Subsequent studies demonstrated that an activated form of PI 3-kinase restored Rac GTP loading in the presence of EGFR inhibition, while a dominant-negative form of PI 3-kinase blocked Rac GTP loading in fibronectin-adherent cells. Our further functional studies identified Vav2, a known exchange factor for Rac, as a crucial downstream component in EGFRand PI 3-kinase-dependent Rac activation upon integrinmediated cell adhesion. Our results provide a mechanistic insight into integrin-dependent Rac activation, and identify a novel role for EGFR, PI 3-kinase and Vav2 in this pathway.

BackgroundCongenital dyserythropoietic anemia type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with internuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of congenital dyserythropoietic anemia type I erythroblasts and data on a role in cell cycle progression of codanin-1 homolog in Drosophila we investigated the cellular localization and possible involvement of codanin-1 during the cell cycle. Design and MethodsCodanin-1 localization was studied by immunofluorescence and immune electron microscopy. Cell cycle expression of codanin-1 was evaluated using synchronized HeLa cells. E2F proteins are the main regulator of G1/S transition. An E2F1-inducible cell line (U20S-ER-E2F1) enabled us to study codanin-1 expression following ectopic E2F1 induction. Direct binding of E2F1 to codanin-1 promoter was assessed by chromatin immunoprecipitation. We used a luciferase-reporter plasmid to study activation of CDAN1 transcription by E2F1. ResultsWe localized codanin-1 to heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in the S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with its exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, containing five putative E2F binding sites, was found to be a direct target of E2F1. ConclusionsTaken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in the S phase. The exact role of codanin-1 during the S phase remains to be determined. Nevertheless this represents the first step towards understanding the function of the proteins involved in congenital dyserythropoietic anemia.Key words: codanin-1, cell cycle, heterochromatin, E2F.Citation: Dgany O, Lahmi R, Marcoux N, Krasnov T, Yissachar N, Ginsberg D, Motro B, Resnitzky P, Yaniv I, Kupfer GM, the ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n S. Noy-Lotan et al.

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SummaryProfilin-deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1⌬ mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1⌬ cells mate as well as wild-type cells, while the mating efficiency of MAT␣, pfy1⌬ cells is reduced. Pulse-chase experiments demonstrate an accumulation of the 19 kDa ␣-factor precursor and delayed secretion of the mature ␣-factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the ␣-factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1⌬ cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high-copy-number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, ␣-factor maturation and the accumulation of intracellular membranous structures in pfy1⌬ cells.