Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions.

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.

Keap1 regulates the oxidation-sensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism.

Keap1 is a negative regulator of Nrf2, a transcription factor essential for antioxidant response element (ARE)-mediated gene expression. We find that Keap1 sequesters Nrf2 in the cytoplasm, not by docking it to the actin cytoskeleton but instead through an active Crm1/exportin-dependent nuclear export mechanism. Deletion and mutagenesis studies identified a nuclear export signal (NES) in the intervening region of Keap1 comprised of hydrophobic leucine and isoleucine residues in agreement with a traditional NES consensus sequence. Mutation of the hydrophobic amino acids resulted in nuclear accumulation of both Keap1 and Nrf2, as did treatment with the drug leptomycin B, which inactivates Crm1/exportin. ARE genes were partially activated under these conditions, suggesting that additional oxidation-sensitive elements are required for full activation of the antioxidant response. Based on these data, we propose a new model for regulation of Nrf2 by Keap1. Under normal conditions, Keap1 and Nrf2 are complexed in the cytoplasm where they are targeted for degradation. Oxidative stress inactivates Keap1's NES, allowing entry of both Keap1 and Nrf2 into the nucleus and transcriptional transactivation of ARE genes.

Keap1 in adhesion complexes.

Cell adhesion complexes are sensors that interact with the extracellular environment and allow for the transmission of signals found outside the cell across the plasma membrane to the cell interior. Keap1 is a newly identified component of cell adhesion complexes. We investigated Keap1's association with these complexes in diverse tissues and cell types. Keap1 is present in focal adhesion (FA)-like assemblies in kidney proximal tubule cells where it colocates with actin. In liver, Keap1 is found in the adherens junctions (AJ) and at the base of the bile canaliculi. To study Keap1's involvement in both the integrin-based FA and the cadherin-based AJ, we induced formation of these complexes in fibroblasts, using a serum starvation followed by a serum supplementation method. When compared with vinculin, a component of all FA, we found that Keap1 assembles only in the peripheral FA. Within the peripheral FA, Keap1 was present in distinct foci along the length of the FA and these foci were different from vinculin, talin, paxillin, and phospho-tyrosine rich regions of the FA. Unlike most FA components, Keap1 was also recruited to the newly formed AJ. As Keap1 homologues are actin-bundling proteins, we hypothesize that Keap1's function is to bundle F-actin within these diverse types of cell adhesion components.

A human homologue of Drosophila kelch associates with myosin-VIIa in specialized adhesion junctions.

Mutations in myosin-VIIa are responsible for the deaf-blindness, Usher disease. Myosin-VIIa is also highly expressed in testis, where it is associated with specialized adhesion plaques termed ectoplasmic specializations (ES) that form between Sertoli cells and germ cells. To identify new roles for myosin-VIIa, we undertook a yeast two-hybrid screen to identify proteins associated with myosin-VIIa in the ES. We identified Keap1, a human homologue of the Drosophila ring canal protein, kelch. The kelch-repeats in the C-terminus of human Keap1 associate with the SH3 domain of myosin-VIIa. Immunolocalization studies revealed that Keap1 is present with myosin-VIIa in the actin bundles of the ES. Myosin-VIIa and Keap1 copurify with ES and colocate with each other and with F-actin at the electron microscopy level. Interestingly, in many epithelial cell types including cells derived from retina and inner ear, Keap1 is a component of focal adhesions and zipper junctions. Keap1 can target to the ES in the absence of myosin-VIIa, suggesting that Keap1 associates with other molecules in the adhesion plaque. Keap1 and myosin-VIIa overlapped in expression in the inner hair cells of the cochlea, suggesting that Keap1 may be a part of a family of actin-binding proteins that could be important for myosin-VIIa function in testis and inner ear.