Chp, a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton.

The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.

The ras recruitment system, a novel approach to the study of protein-protein interactions.

The yeast two-hybrid system represents one of the most efficient approaches currently available for identifying and characterizing protein-protein interactions [1-4]. Although very powerful, this procedure exhibits several problems and inherent limitations [5]. A new system, the Sos recruitment system (SRS), was developed recently [6] based on a different readout from that of the two-hybrid system [6-8]. SRS overcomes several of the limitations of the two-hybrid system and thus serves as an attractive alternative for studying protein-protein interactions between known and novel proteins. Nevertheless, we encountered a number of problems using SRS and so have developed an improved protein recruitment system, designated the Ras recruitment system (RRS), based on the absolute requirement that Ras be localized to the plasma membrane for its function [9-10]. Ras membrane localization and activation can be achieved through interaction between two hybrid proteins. We have demonstrated the effectiveness of the novel RRS system using five different known protein-protein interactions and have identified two previously unknown protein-protein interactions through a library screening protocol. The first interaction (detailed here) is between JDP2, a member of the basic leucine zipper (bZIP) family, and C/EBPgamma, a member of the CCAAT/enhancer-binding protein (C/EBP) family. The second interaction is between the p21-activated protein kinase Pak65 and a small G protein (described in the accompanying paper by Aronheim et al. [11]). The RRS system significantly extends the usefulness of the previously described SRS system and overcomes several of its limitations.

Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells.

Here, we describe an application of the backbone cyclic (BC) proteinomimetic approach to the design and the synthesis of a BC peptide which functionally mimics the nuclear localization signal (NLS) region of the human immunodeficiency virus type 1 matrix protein (HIV-1 MA). On the basis of the NMR structure of HIV-1 MA, a library of BC peptides was designed and screened for the ability to inhibit nuclear import of NLS-BSA in digitonin-permeabilized HeLa and Colo-205 cultured cells. The screening yielded a lead compound (IC50 = 3 microM) which was used for the design of a second library. This library led to the discovery of a highly potent BC peptide, designated BCvir, with an IC50 value of 35 nM. This inhibitory potency is compared to a value of 12 microM exhibited by the linear parent HIV-1 MA NLS peptide. BCvir also reduced HIV-1 production by 75% in infected nondividing cultured human T-cells and was relatively resistant to tryptic digestion. These properties make BCvir a potential candidate for the development of a novel class of antiviral drugs which will be based on blocking nuclear import of viral genomes.

Translocation of NLS-BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract. An experimental system for studying plant cytosolic factors involved in nuclear import.

An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)-containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS-containing proteins into nuclei of permeabilized HeLa cells was demonstrated. The results described in the present work show that nuclear import of fluorescently labeled BSA conjugates bearing the NLS sequence of SV40 large T antigen could be supported by petunia cell cytoplasmic extract. This heterologous system shows the characteristic features of the homologous mammalian system, namely, is ATP dependent and is inhibited by WGA, GTPgammaS as well as by non-fluorescent NLS-BSA conjugates. The system described here offers an experimental method to study and characterise cytosolic factors which are required for nuclear import in plants.