T cell receptor (TCR) stimulation activates the small GTPase Rap1A, which is reported to antagonize Ras signaling and induces T cell anergy. To address its role in vivo, we generated transgenic mice that constitutively expressed active Rap1A within the T cell lineage. We found that active Rap1A did not interfere with the Ras signaling pathway or antagonize T cell activation. Instead of anergy, the T lymphocytes that constitutively expressed active Rap1A showed enhanced TCR-mediated responses, both in thymocytes and mature T cells. In addition, Rap1A activation was sufficient to induce strong activation of the beta1 and beta2 integrins via an avidity-modulation mechanism. This shows that, far from playing an inhibitory role during T cell activation, Rap1A positively influences T cells by augmenting lymphocyte responses and directing integrin activation.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) ) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser 748 in PKD (pS748

The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences. The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis. Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract. Six polypeptides (ϳ110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p. Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p. Microsequencing of the 43-and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively. Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits. Human ORC5 was also cloned; its sequence displayed extensive homology to both Drosophila and yeast ORC5. Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p. Finally, we present the sequence of the putative Xenopus and human Orc3p.The initiation of DNA replication in eukaryotes is tightly regulated such that all the DNA is replicated precisely once per cell cycle from a number of discrete replication origins (Refs. 1-3; for reviews, see Refs. 4 and 5). The nature of this regulation, however, is not well understood as only a small fraction of proteins involved in this process have been identified. Moreover, with the exception of budding yeast, the DNA sequences that define the origin of DNA replication on chromosomes remain an almost complete mystery in higher eukaryotes.Present knowledge of the control over initiation of DNA replication in an eukaryotic cell is largely based on experiments done in the yeast Saccharomyces cerevisiae. In this yeast, DNA replication starts from well defined origins, known as the autonomously replicating sequences (6). An essential, bipartite DNA sequence in all S. cerevisiae replication origins is recognized by the six-subunit origin recognition complex (ORC), 1 which is itself essential for initiation of DNA replication (7-20). The ORC also functions in the control of gene silencing, a function that is, at least partially, separable from the functions in initiation of DNA replication (9, 10, 15, 21-23). Proteins homologous to individual subunits of the S. cerevisiae ORC have been identified recently in other yeast species and from animal and plant cells. Orc1p and Orc2p were found in Schizosaccharomyces pombe, Drosophila, Xenopus, and humans, and Orc5p has been identified in Drosophila (24 -30). More recently, the human Orc4p has been identified and shown to co-immunoprecipitate with human Orc2p (31). Immunodepletion of ORC inhibited initiation of DNA replication in a Xenopus egg extract, sugge...