Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of 1 integrin

Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R?/? mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R?/? keratinocytes, and 4.1R?/? mice exhibited defective epidermal wound healing. Cultured 4.1R?/? keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of ?1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of ?1 integrin, possibly through a direct association between 4.1R and ?1 integrin

PtdIns(4,5)P-restricted plasma membrane localization of FAN is involved in TNF-induced actin reorganization

The WD-repeat protein factor associated with nSMase activity (FAN) is a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades. However, the precise functional involvement of FAN in specific cellular TNF responses remain unclear. Here, we report the involvement of FAN in TNF-induced actin reorganization and filopodia formation mediated by activation of Cdc42. The pleckstrin-homology (PH) domain of FAN specifically binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P), which targets FAN to the plasma membrane. Site-specific mutagenesis revealed that the ability of FAN to mediate filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding motif, or by the disruption of intramolecular interactions between the PH domain and the adjacent beige and Chediak-Higashi (BEACH) domain. Furthermore, FAN was shown to interact with the actin cytoskeleton in TNF-stimulated cells via direct filamentous actin (F-actin) binding. The results of this study suggest that PH-mediated plasma membrane targeting of FAN is critically involved in TNF-induced Cdc42 activation and cytoskeleton reorganization