Kir, a novel Ras-family G-protein, induces invasive pseudohyphal growth in Saccharomyces cerevisiae.

Kir belongs to a novel class of Ras-family G-proteins which includes Gem and Rad. These proteins are unique among Ras super-family G-proteins since their expression is under transcriptional regulation in mammalian cells. To gain insight into the function of Kir, we took advantage of the well-defined signal transduction pathways of yeast. When kir is expressed in Saccharomyces cerevisiae, the transformants form pseudohyphae and exhibit invasive properties characteristics of yeast cells undergoing a developmental transition induced by nitrogen starvation. Analysis of pseudohyphal signaling pathway mutants suggests that the Kir-induced pseudohyphae formation requires a MAP kinase cascade involving ste20, ste11, ste7 but not ste5 gene products. Furthermore, our results are consistent with the idea that Kir functions upstream of the STE20 kinase which plays a critical role in two distinct MAP kinase cascades.

Endocytosis and intracellular degradation of heterologous protein by eosinophilic granulocytes isolated from rainbow trout (Oncorhynchus mykiss) posterior intestine.

Adherence capacity to tissue substrate, endocytosis capacity for heterologous and homologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finally, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue.

Several Plasmodium falciparum genes encoding cdc2-related protein kinases have been identified, but the modalities of their regulation remains largely unexplored. In the present study, we investigated the regulation in vitro of PfPK5, a putative homologue of Cdk1 (cdc2) in P. falciparum. We show that (i) PfPK5 is efficiently activated by heterologous (human) cyclin H and p25, a cyclin-like molecule that specifically activates human Cdk5; (ii) the activated enzyme can be inhibited by chemical Cdk inhibitors; (iii) Pfmrk, a putative P. falciparum homologue of the Cdk-activating kinase, does neither activate nor phosphorylate PfPK5; and (iv) PfPK5 is able to autophosphorylate in the presence of a cyclin. Taken together, these results suggest that the regulation of Plasmodium Cdks may differ in important aspects from that of their human counterparts. Furthermore, we cloned an open reading frame encoding a novel P. falciparum protein possessing maximal homology to cyclin H from various organisms, and we show that this protein, called Pfcyc-1, is able to activate recombinant PfPK5 in vitro with an efficiency similar to that of human cyclin H and p25. This work opens the way to the development of screening procedures aimed at identifying compounds that specifically target the parasite Cdks.

An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature.

The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli. The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms. The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts. Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity. Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector. Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity. Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location. This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2. The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting.

Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.

Transcriptional activation of a ras-like gene (kir) by oncogenic tyrosine kinases.

We report the characterization of a member of the ras gene family that is overexpressed in cells transformed by abl tyrosine kinase oncogenes. The gene, named kir (for kinase-inducible ras-like), is induced at the transcriptional level. kir mRNA has a rapid turnover and encodes a protein of 33 kDa with guanine nucleotide-binding activity but undetectable intrinsic GTPase activity. kir was cloned by differential screening of genes present in fully malignant versus growth factor-independent cell lines expressing wild-type or mutant forms of BCR/ABL. BCR/ABL and v-Abl induce transcription of the kir gene via specific signaling pathway(s), but kir overexpression alone is not sufficient to mediate transformation.

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.