Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca2+-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acidsensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the twochain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.

Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-kappaB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36-expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.