Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.
The cellular response to DNA damage is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 protein phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. To investigate DNA damage responses mediated by the p38/stress-activated protein kinase (SAPK) axis of signaling, the optimal phosphorylation motifs of mammalian p38alpha SAPK and MAPKAP kinase-2 were determined. The optimal substrate motif for MAPKAP kinase-2, but not for p38 SAPK, closely matches the 14-3-3 binding site on Cdc25B/C. We show that MAPKAP kinase-2 is directly responsible for Cdc25B/C phosphorylation and 14-3-3 binding in vitro and in response to UV-induced DNA damage within mammalian cells. Downregulation of MAPKAP kinase-2 eliminates DNA damage-induced G2/M, G1, and intra S phase checkpoints. We propose that MAPKAP kinase-2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells.
Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type. Using this system, we biochemically dissect molecular events and signaling pathways involved in NADPH oxidase assembly and demonstrate specific roles for PKC delta, PI(3,4)P(2)/PI(3,4,5)P(3), and PI(3)P in the PMA-dependent intracellular activation process. This system should be of great utility for the study of cell signaling events that regulate the intracellular production of reactive oxygen species by neutrophils.
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