MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells.

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.

The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.

NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.

An insulinoma nuclear factor binding to GGGCCC motifs in human insulin gene.

Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on 5' flanking DNA elements. In the enhancer of rat I insulin gene, two elements, the Nir and Far boxes, located at positions -104 and -233 respectively and containing the same octameric motif are essential for B cell specific transcription activity. Homologous sequences are present in the human insulin gene. While studying the binding of nuclear proteins from insulinoma cells to the -258/+241 region of the human insulin gene, we observed a previously undetected protein binding site in the intron I region between nucleotides +160 and +175. The binding activity was present in insulin producing cells such as RIN and HIT insulinoma cells but not in fibroblasts or insulin negative fibroblast x RIN hybrid cells. DNAse I footprinting and gel retardation/methylation interference experiments allowed us to define the core binding site of the intron binding factor as a GGGCCC hexamer. This factor is also capable to bind to a related sequence, contiguous to the Far-like element in rat and human insulin genes. The binding of the GGGCCC binding factor in this critical region of the insulin gene enhancer may participate in the regulation of insulin gene expression.

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation.

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.

Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56).

Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56.

Detection of tyrosine-specific protein kinases with gastrin as exogenous substrate.

Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent Km is 65 microM and the maximum rate 1900 pmol/min per mg; the kinase is more efficient with MnCl2 than with MgCl2, is stimulated by NaVO3 and inhibited by ZnCl2. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells: human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cells: human T and B lymphocytes.

Acute myeloblastic leukemia with active tyrosine protein kinase.

High level of tyrosine protein kinase activity was found in a membrane fraction isolated from an acute myeloblastic leukemia, out of 24 leukemias of different origin investigated. The major substrate for tyrosine phosphorylation in vitro was a 58 kDA protein (p58). The phosphorylation proceeded actively at 0 degrees C and was strongly stimulated by Mn2+ ions. Comparison by partial proteolysis of the p58 with similar phosphoproteins from a T-lymphoma line (KE37) and from lectin stimulated lymphocytes showed high similarity. The possible role of the tyrosine kinase activity in this leukemia is discussed.

Identification of a 58,000 daltons phosphoprotein with tyrosine protein kinase activity in a murine lymphoma cell line.

A very high level of tyrosine protein kinase (TPK) activity has been recently detected in a murine lymphoma, induced by Moloney murine leukemia virus. A major endogenous substrate for tyrosine phosphorylation in vitro is a protein of Mr 55-60,000 (p58) associated with the detergent insoluble matrix of LSTRA cells; in the present work p58 was solubilized, isolated by anion exchange chromatography and then precipitated by antiphosphotyrosine antibodies. Through these steps of isolation, TPK activity was measured by the use of a simplified gastrin phosphorylation assay. It is demonstrated that the TPK activity copurifies with p58, which leads to the conclusion that p58 bears itself the enzymatic activity. Although functionally similar to other enzymes of this group, this newly characterized TPK does not seem to be closely related to one of the previously documented TPK. This suggests either that this protein is the product of a so far unrecognized cellular TPK gene or that it derives from a rearrangement of one of the previously described TPK genes.

The rate of initiation of alpha- and beta-globin mRNA translation is modulated by 50 kDa, 28-kDa and 24-kDa polypeptide-containing fractions.

The relative rates of initiation of alpha- and beta-globin mRNA translation in a Krebs II ascites cell-free system are differently modulated by a 50-kDa protein and two fractions containing either a 28-kDa or a 24-kDa polypeptide. Each of these fractions stimulated a discrete step that limits initiation of protein synthesis, but other rate-limiting steps take place upstream and/or downstream, resulting in characteristic kinetics of the stimulation of alpha- and beta-globin synthesis. The ascites extracts appear to be deficient in these activities.