It seemed, however, better to work it on native rather than denatured proteins

It seemed, however, better to work it on native rather than denatured proteins. Indeed probably because of the conformation of the prenyl group on nitrocellulose, it did not exhibit good sensitivity in Western blot, whereas it ICI-118551 worked very well in immunoprecipitation. restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB Mouse monoclonal antibody to Keratin 7. The protein encoded by this gene is a member of the keratin gene family. The type IIcytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratified epithelial tissues. This type IIcytokeratin is specifically expressed in the simple epithelia lining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding the type II cytokeratinsare clustered in a region of chromosome 12q12-q13. Alternative splicing may result in severaltranscript variants; however, not all variants have been fully described from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is usually farnesylated farnesylated or geranylgeranylated either ICI-118551 by FTase or by GGTase I, respectively (23). Comparable results were obtained for the Ras-related protein TC21, whose C terminus CVIF would predict geranylgeranylation (24). Moreover, when cells are treated with farnesyltransferase inhibitors (27) and (28). These reports suggest that the determinants of enzyme recognition appear to be more complex than initially thought, especially for GGTase I. Nonetheless, it is still little comprehended how K-Ras4B or RhoB can be alternatively prenylated. It was hypothesized that this sequence responsible in RhoB and in KRas-4B should be around the NH2-terminal extremity of the CAAX box or attributable in part to the polylysine sequence immediately upstream of the CAAX box, respectively (29, 30). Recently Du (31) showed that this substitution of the last 16 carboxyl-terminal residues of RhoB with the ICI-118551 13 carboxyl-terminal residues of RhoA directs the solely geranylgeranylation of RhoB, and proposed that geranylgeranylated RhoB might have a biological role distinct from that of farnesylated RhoB (28, 31). The specific prenylation of the proteins is usually of particular interest in view of the importance of functional consequences of prenylation. Prenylation is not only necessary for membrane association but also for proteinCprotein interactions (5), and the nature of the linked isoprenoid can influence protein interactions, such as for trimeric G proteins and receptor (32). To further investigate the functional consequence of simply adding different types of prenyl groups to RhoB without affecting the sequence of the mature protein, we analyzed the effect of solely substituting the CAAX box on prenylation of the RhoB protein. To characterize the added prenyl group around the mutated protein, we used not only the baculovirus system that had been demonstrated to be efficient for prenylation analysis (33) but also developed an anti-farnesyl polyclonal antibody that could recognize the farnesylated form not only of RhoB but also of H-Ras. Materials and Methods Plasmid Constructions. Standard PCR mutagenesis techniques were used to generate plasmids coding for RhoB with the wild type (RhoB-CKVL), with a farnesylated (RhoB-CAIM), or a geranylgeranylated (RhoB-CLLL) CAAX sequence, or not prenylated (RhoB). PCR amplifications of RhoB from pCB6-VSV-RhoB (a generous gift of B. Olofsson, Centre National de la Recherche Scientifique, Gif/Yvette, France) were done with the 5 primer: GAAGATCTGGTACCATGGCGGCCATCCGC and the 3 primers: CGGGATCCTCATAGCACCTTGCAGCA (RhoB-CKVL), CGGGATCCTCACATGATGGCGCAGCAGTTGATGCAGCCGTTCAG (RhoB-CAIM), CGGGATCCTCACAGCAGCAGGCAGCAGTTGATGCAGCCGTTCTG (RhoB-CLLL), or CGGGATCCTCAGCAGTTGATGCAGCCGTTCAG (RhoB), respectively. The amplified fragments ICI-118551 digested by (35). (36). The 1H and 13C spectra confirmed that alkylation occurred at the sulfhydryl moiety. MS (DCI/NH3, CHCl3): 340 (MH+). 13CNMR (50 MHz, CDCl3): (ppm): 29.9 ( cysteinyl carbon), 36.4 (C1 farnesyl). (28) and to have distinct biological functions according to its prenylation. To characterize the functional consequence of this differential prenylation of RhoB, we ICI-118551 generated mutants of the protein by altering the CAAX sequence to produce specific prenylation. The CKVL sequence of RhoB (Rho-CKVL) was replaced by standard PCR mutagenesis either by the CAAX box of lamin B (RhoB-CAIM), Rap1A (RhoB-CLLL), or deleted (RhoB-). We showed in a parallel work using the RhoB mutants that geranylgeranylated and farnesylated forms of RhoB appeared to have similar functions in human cells in contrast with the rodent cells (38). The baculovirus expression system was first used to determine whether the nature of the CAAX motif influenced the specificity of the RhoB prenylation. Expression of the different His-tagged RhoB forms in Sf9 insect cells was confirmed by Western blot after purification using Ni2+ affinity chromatography. As shown in Fig. ?Fig.1,1, the major band obtained after elution with 250 mM imidazole, detected by SDS/PAGE and silver staining, corresponded to RhoB protein that was expressed, whatever the contamination, with similar rates. Open in a separate window Physique 1 The CAAX box.