CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X) 6 P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. CD2-associated protein (CD2AP)2 is a 70-kDa protein that was originally cloned as a protein that interacts with the cytoplasmic tail of CD2, a T lymphocyte and natural killer cell transmembrane protein (1). It is composed of three Src homology 3 (SH3) domains at the NH 2 terminus followed by proline-rich sequences and a coiled-coil domain at the extreme COOH terminus. It is expressed in all tissues except brain. Interestingly, CD2AP-deficient animals die of renal failure ϳ6 weeks of age (2). In the kidney, CD2AP is highly expressed in the glomerular epithelial cell, and it is implicated to play a role in a specialized cell junction known as a slit diaphragm (3).A homolog of CD2AP, Cin85, was cloned as an interacting protein with the E3 ubiquitin ligase c-cbl (4) and as an inhibitor of phosphatidylinositol 3-kinase (5). Recently, several endocytic and actin-associated molecules have been reported to interact with CD2AP and Cin85. Some proteins have been demonstrated to interact with both CD2AP and Cin85, whereas others have only been shown to bind one or the other. CD2AP has been shown to play a role in vesicular trafficking because of interactions with c-cbl and an active form of Rab4 (6). Cin85 has also been shown to bind to molecules involved in endocytosis, such as endophilin, synaptojanin 2B1, and SHIP-1 and the clathrin scaffold HIP1R (7,8). Both CD2AP and Cin85 contain a motif, FXDXF, that mediates interactions with the ␣-appendage of clathrin adaptor protein 2 (9). Interactions of CD2AP and Cin85 with the phosphatidylinositol bisphosphate-dependent GTPase for ARF1 and ARF5, known as ASAP1, as well as cortactin-and actin-capping protein suggest additional roles in the regulation of the actin cytoskeleton (7, 10 -12).To further elucidate the molecular mechanism of CD2AP function, we performed mass spectrometry to identify interacting proteins. We identified novel and previously known interacting proteins such as actin-capping protein CP (11).Over the last decade, there has been much progress in our understanding of how the actin cytoskeleton is regulated. Critical is the polymerization of monomeric G-actin to forming an asymmetric actin filament with a barbed and a poin...

Understanding the physiology of complex relationships between components of signaling pathways and the actin cytoskeleton is an important challenge. CD2AP is a membrane scaffold protein implicated in a variety of physiological and disease processes. The physiological function of CD2AP is unclear, but its biochemical interactions suggest that it has a role in dynamic actin assembly. Here, we report that CD2AP functions to facilitate the recruitment of actin capping protein (CP) to the Src kinase substrate, cortactin, at the cell periphery, and that this is necessary for formation of the short branched filaments that characterize lamellipodium formation and are required for cell migration. Superresolution fluorescence microscopy demonstrated that the efficient colocalization of CP and cortactin at the cell periphery required CD2AP. As both cortactin and CP function to enhance branched actin filament formation, CD2AP functions synergistically to enhance the function of both proteins. Our data demonstrate how the interplay between specialized actin regulatory molecules shapes the actin cytoskeleton.

The genome of the pathogenic bacterium Listeria monocytogenes contains a family of genes encoding proteins with a leucine-rich repeat domain. One of these genes, inlH, is a B -dependent virulence gene of unknown function. Previously, inlH was proposed to be coexpressed with two adjacent internalin genes, inlG and inlE. Using tiling arrays, we showed that inlH expression is monocistronic and specifically induced in stationary phase as well as in the intestinal lumen of mice, independent of inlG and inlE expression. Consistent with inlH B -dependent regulation, surface expression of the InlH protein is induced when bacteria are subjected to thermal, acidic, osmotic, or oxidative stress. Disruption of inlH increases the amount of the invasion protein InlA without changing inlA transcript level, suggesting that there is a link between inlH expression and inlA posttranscriptional regulation. However, in contrast to InlA, InlH does not contribute to bacterial invasion of cultured cells in vitro or of intestinal cells in vivo. Strikingly, the reduced virulence of inlH-deficient L. monocytogenes strains is accompanied by enhanced production of interleukin-6 (IL-6) in infected tissues during the systemic phase of murine listeriosis but not by enhanced production of any other inflammatory cytokine tested. Since InlH does not modulate IL-6 secretion in macrophages at least in vitro, it may play a role in other immune cells or contribute to a pathway that modulates survival or activation of IL-6-secreting cells. These results strongly suggest that InlH is a stress-induced surface protein that facilitates pathogen survival in tissues by tempering the inflammatory response.

Many virulence factors of Gram-positive bacterial pathogens are covalently anchored to the peptidoglycan (PG) by sortase enzymes. However, for rod-shaped bacteria little is known about the spatiotemporal organization of these surface proteins in the cell wall. Here we report the three-dimensional (3D) localization of the PG-bound virulence factors InlA, InlH, InlJ, and SvpA in the envelope of Listeria monocytogenes under different growth conditions. We found that all PG-anchored proteins are positioned along the lateral cell wall in nonoverlapping helices. However, these surface proteins can also become localized at the pole and asymmetrically distributed when specific regulatory pathways are activated. InlA and InlJ are enriched at poles when expressed at high levels in exponential-phase bacteria. InlA and InlH, which are B dependent, specifically relocalize to the septal cell wall and subsequently to the new pole in cells entering stationary phase. The accumulation of InlA and InlH in the septal region also occurs when oxidative stress impairs bacterial growth. In contrast, the iron-dependent protein SvpA is present at the old pole and is excluded from the septum and new pole of bacteria grown under low-iron conditions. We conclude that L. monocytogenes rapidly reorganizes the spatial localization of its PG proteins in response to changes in environmental conditions such as nutrient deprivation or other stresses. This dynamic control would distribute virulence factors at specific sites during the infectious process.