Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search

Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. provide important control of Rap activation. This is particularly relevant for Rap1, one of the Rap family members that is present on both the plasma membrane (PM) and the nuclear envelope (NE) (4). Through specific protein interactions, properly localized Epac proteins may not only function more efficiently but also limit the activation of Rap1 to specific regions within the cell. Direct support for this model comes from detailed structural and functional analysis of Epac proteins in the context of a cell. Both Epac proteins contain homologous regulatory and catalytic regions. Direct binding of cAMP to each regulatory region triggers a conformational change that allows its respective catalytic region to activate Rap1. The catalytic regions of both Epac1 and Epac2 comprise a Ras exchanger motif (REM) domain and a CDC25 homology domain that confers GEF activity toward Rap1. For Epac2, PR-104 the REM and CDC25 domains are interrupted by a Ras association (RA) domain that interacts with activated, GTP-loaded Ras at an affinity similar to those of the classical Ras effectors, such as Raf-1 and B-Raf (28). Because the locations of Ras and Rap1 can overlap at the plasma membrane, the recruitment of Epac2 to Ras brings it into proximity with a pool of Rap1 at this locale (27). This compartmentalization of Epac2 allows it to activate Rap1 at the PM efficiently in the presence PR-104 of cAMP. In contrast to the findings for Epac2, the region between the REM and CDC25 domains in Epac1 contains a putative RA domain for which no binding partner has been identified. Unlike Epac2, Epac1 is predominantly localized to the perinuclear region instead of the PM in multiple cell lines (12, 32, 41, 49). Recent evidence suggests that Epac1 may function in nuclear processes, such as the nuclear transport of DNA-dependent protein kinase (DNA-PK) (21). However, the mechanism for the spatial regulation of Epac1-Rap1 signaling in this subcellular location is unknown. In this study, we identify a novel mechanism underlying the anchored signaling of Epac1 at the nuclear pore via its putative RA domain, and we report for the first time the coupling of the nuclear small GTPase Ran and Rap1 at the NE. MATERIALS AND METHODS Plasmids. Human Epac1 was a gift from Johannes Bos, Utrecht University. Hemagglutinin (HA)-tagged wild-type Ran and RanV19 were gifts from Ian Macara, University of Virginia. Histone 2B-mCherry was obtained from Addgene (Cambridge, MA). GFP-Epac1, Epac2, Flag-Rap1b, and mCherry-RasV12 have been described previously (28, 49). HRas and Rap2b cDNAs were purchased from the Missouri Rabbit Polyclonal to MSK2 S&T cDNA Resource Center (Rolla, MO) PR-104 and were subcloned into the pGFP-C1 and/or pmCherry-C1 vector (Clontech, Mountain View, CA). Epac1RA2 was constructed by inserting a PCR product for amino acids (aa) 559 to 720 of Epac2 between the sequences for aa 423 and 586 of Epac1 and was subcloned into the pcDNA3 and pGFP-C1 vectors. GFP-Epac1295 was constructed by subcloning the PCR product for aa 295 to 881 of Epac1 into pGFP-C1. GFP-Epac1673 was constructed by digestion using existing BglII and PstI sites in GFP-Epac1295 and ligation of the blunted ends. GFP-Epac1295RA2 was constructed by subcloning the PCR product for aa.