Listeria monocytogenes is a Gram-positive, food-borne bacterial pathogen capable of causing gastroenteritis, meningitis, or abortions (39, 32). Listeria induces its own internalization (entry) into nonphagocytic mammalian cells, a process that likely plays an important role in traversal of the intestinal, placental, and blood-brain barriers (7,14,16,23). One of the pathways of Listeria entry is mediated by interaction of the bacterial surface protein InlB with its host receptor, the Met receptor tyrosine kinase (16, 37).InlB-Met interaction triggers activation (tyrosine phosphorylation) of the Met receptor and subsequent rearrangements in the F-actin cytoskeleton of the mammalian cell (16,29). These cytoskeletal changes remodel the host cell surface, resulting in engulfment of adherent Listeria. One of the host mammalian proteins that acts downstream of Met to promote F-actin rearrangements and bacterial entry is type IA phosphoinositide (PI) 3-kinase (8,17,18). This PI 3-kinase is a heterodimeric enzyme comprised of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (4, 11). p85-p110 generates phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 )] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], which are lipid second messengers that regulate a variety of biological processes, including growth, survival, and motility of mammalian cells. A plethora of downstream "target" proteins that bind PI(4,5)P 2 and/or PI(3,4,5)P 3 and mediate the biological effects of p85-p110 have been identified (4, 24). However, mammalian proteins that act downstream of type IA PI 3-kinase to control Listeria uptake have yet to found.In this work, we demonstrate that the human GTPase-activating protein (GAP) ARAP2 is required for InlB-mediated cytoskeletal changes and entry of Listeria. ARAP2 is known to bind PI(3,4,5)P 3 , resulting in upregulation of a GAP domain that inactivates the mammalian GTPase Arf6 (42). We provide genetic evidence indicating that the ArfGAP domain of ARAP2 stimulates Listeria entry by antagonizing Arf6. ARAP2 has several functional domains in addition to its ArfGAP domain. One of these domains in ARAP2 interacts with the mammalian GTPase RhoA (42). Our genetic data demonstrate that this "RhoA binding" (RB) domain also plays a critical role in bacterial entry. Surprisingly, pharmacological experiments indicate that the RB domain controls Listeria uptake through an unknown mechanism that does not involve Rho proteins. Our work indicates a key role for host ARAP2 in InlB-mediated entry of Listeria. One of the likely ways that type IA PI 3-kinase controls entry of Listeria is through regulation of ARAP2. MATERIALS AND METHODSBacterial strains, mammalian cell lines, and media. The wild-type Listeria monocytogenes strain EGD and the isogenic ⌬inlB mutant strain containing an in-frame deletion of the inlB gene were previously described (10). These strains were grown in brain heart infusion (BHI) broth (Difco) and prepared for infection as described previously (10,18).The human epithelial cell lin...

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Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S3 resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.

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