UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway.

Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.

The PH-PxxP domain of RalGPS2 promotes PC12 cells differentiation acting as a dominant negative for RalA GTPase activation.

RalGPS2 is a guanine nucleotide exchange factor for RalA GTPase characterized by a C-terminal Pleckstrin Homology (PH) domain; this GEF is endogenously expressed in PC12 cells and in rat brain but its role in PC12 cells and in cell differentiation is actually unknown. Here we have shown that transient expression of RalGPS2-PH-PxxP domain in PC12 and PC12-TrkA cells induces high level of neurite outgrowth; this differentiation is comparable with that of PC12 cells treated with RalGPS2 siRNA. Stable PC12 cell lines expressing the PH-PxxP domain of RalGPS2 have been generated; in these cell lines the PH-PxxP domain acts as a dominant negative for RalA activation, promotes cells differentiation and re-directs NGF signals towards MAPKs. Furthermore it has been also demonstrated that the PH-PxxP domain of RalGPS2 induces microspikes formation a typical feature of cells in which the Cdc42 GTPase is constitutively activated.

RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX.

BACKGROUND: Modulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processes that govern gene silencing, either by inhibiting or activating transcription, and maintain genome stability, acting as scaffold to properly organize the DNA damage response. Thus, it is of paramount importance the identification and the characterization of new ubiquitin ligases that address histones. RESULTS: We identified and characterized RNF168, a new chromatin-associated RING finger protein. We demonstrated that RNF168 is endowed with ubiquitin ligase activity both in vitro and in vivo, which targets histones H2A and H2AX, but not H2B, forming K63 polyubiquitin chains. We previously described the presence within RNF168 sequence of two MIU domains, responsible for the binding to ubiquitinated proteins. Here we showed that inactivation of the MIUs impairs ubiquitin binding ability in vitro and reduces chromatin association of RNF168 in vivo. Moreover, upon formation of DNA double strand breaks induced by chemical and physical agents, RNF168 is recruited to the DNA damage foci, where it co-localizes with gammaH2AX and 53BP1. The localization of RNF168 at the site of damage highly increases the local concentration of ubiquitinated proteins and determines the prolonged ubiquitination signal. CONCLUSION: The RING finger protein RNF168 is a new ubiquitin ligase that functions as chromatin modifier, through histone ubiquitination. We hypothesize a dual function for RNF168. In normal condition RNF168 modifies chromatin structure by modulating ubiquitination of histone H2A. Upon DNA lesions, RNF168 is recruited to DNA damage response foci where it contributes to increase the amount of ubiquitinated proteins, thereby facilitating the downstream signalling cascade.

Functional analysis of RalGPS2, a murine guanine nucleotide exchange factor for RalA GTPase.

RalGPS2 is a murine guanine nucleotide exchange factor of the RalGPS family; it contains a Cdc25-like GEF domain and does not exhibit a Ras-binding domain. The main characteristic of RalGPS2 is its pleckstrin homology (PH) domain, present at the C terminus, that preferentially binds phosphatidylinositol-4,5-biphosphate and in HEK 293 cells localized in membranes, causing ruffling and vesiculation. Moreover, RalGPS2 contains a PxxP motif in the central part of the molecule. This motif binds in vitro and in vivo SH3 domains of Grb2 and PLCgamma. RalGPS2 and its GEF domain activate RalA in vivo while the PH-PxxP domains inhibited it behaving as a dominant negative for the RalA pathway; this activation was not inhibited by co-expression of a dominant negative Ras. RalGPS2 is physiologically expressed in testis and brain; when overexpressed, the whole RalGPS2 causes considerable morphological changes in HEK 293 cells, suggesting its possible role on cytoskeleton reorganization. This is further strengthened by data obtained in NIH3T3 cells where expression of PH-PxxP domain promotes actin depolymerization. Finally, RalGPS2 and its GEF domain induce Ras-independent transcriptional activation of the c-fos promoter in NIH3T3 cells.