p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/ CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helixloop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1. p300 was originally identified by virtue of its ability to bind the viral oncoprotein, ElA (1). A pleiotropic and, likely, important role for p300 in cellular homeostasis is reflected by the fact that its physical association with ElA correlates with the ability of the latter to transform primary cells, to block cellular differentiation along a variety of pathways, and to alter the activity of certain transcriptional enhancer elements (for review, see ref.2). p300 consists of at least two closely related family members: p300 and the CREB-binding protein (CBP) (3). CBP was originally cloned by virtue of its specific binding to the protein kinase A-activated form of CREB (4). Microinjection of CBP antibody blocked the transcriptional activity of CREB, indicating that CBP contributes to CREB function in a major way (5). This observation, along with the ability of p300 to relieve ElA-mediated enhancer suppression, has led to the hypothesis that p300/CBP family members function as specific transcription factor coactivators (6).Remarkably, p300/CBP can interact with other important transcription factors as well. For example, p300/CBP has recently been implicated in the activity of c-fos, c-myb, and MyoD (7,8,33). Thus, it appears that p300/CBP expresses its spectrum of activities, at least in part, by participating in and modulating multiple transcriptional pathways. Therefore, identifying additional p300 interactive proteins should assist in understanding how p300/CBP function contributes to multiple aspects of cellular homeostasis.Nuclear hormone receptors, including retinoic hormone receptor [e.g., retinoic acid receptors (RARs) and retinoic X receptors], thyroid hormone receptors, and steroid hormone receptors (e.g., estrogen receptor), constitute a large family of ligand-dependent transcription factors (9). Although biochemical and genetic studies have revealed much of the molecular detail regarding how nuclear receptors specifically recognize and interact with its target DNA element (10), only recently have workers begun to gain insight into the mechanism underlying ligand activation of nuclear receptor transcription ...

T cell antigen receptor (TCR) stimulation induces the tyrosine phosphorylation of several intracellular proteins including the protooncogene Vav1. Vav1 expression is necessary for normal T cell development and activation. We previously showed that overexpression of Vav1 in Jurkat T cells potentiates the activity of the transcription factor nuclear factor of activated T cells (NF-AT). The mechanism by which Vav1 participates in TCR signaling events is not clear. Vav1 contains a guanine nucleotide exchange factor (GEF) domain that has specificity for Rac and other Rho GTPases that have been recently implicated in T cell activation events. Significantly, in vitro tyrosine phosphoryation of Vav1 by Lck activates its exchange activity. This Lck-mediated phosphorylation of Vav1 has been reported to depend upon Tyr-174 in Vav1, a site implicated in Vav1 function by other studies as well. In this report, we demonstrated that Tyr-174 is not required for the TCR-induced phosphorylation of Vav1 in vivo. Moreover, mutation of Tyr-174 augmented the ability of Vav1 to up-regulate NF-AT activation as well as the Vav1 GEF function leading to Rac activation. However, we also showed that the GEF activity of Vav1 was neither sufficient nor necessary for potentiation of NF-AT, and thereby we identify a GEFindependent role of Vav1 in potentiating NF-AT-driven transcription. Oncogenic Vav1 in which the amino-terminal 67 amino acids were deleted had elevated GEF activity but did not potentiate NF-AT when overexpressed in Jurkat cells. We also showed that a GEF mutant form of Vav1 that had impaired GEF function could still potentiate NF-AT. These studies reveal a previously unrecognized negative regulatory function of Tyr-174 in Vav1 and suggest that domains other than the Vav1 GEF domain contribute to TCR signals leading to NF-AT activation. Engagement of the T cell antigen receptor (TCR)1 with antigen or with cross-linking antibodies initiates a signaling cascade that leads to activation of the T cell. One of the proximal events triggered by TCR engagement is the activation of protein-tyrosine kinases, which results in the tyrosine phosphorylation of several substrates. Previous work has demonstrated that activation of the Src tyrosine kinase Lck is necessary to initiate the signaling cascade. Lck is required to phosphorylate the cytoplasmic tails of the CD3 complex (␥␦⑀) and the chain of the TCR on tyrosines within motifs designated as immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the ITAMs provides docking sites for the Src homology-2 (SH2) domains of the Syk family protein-tyrosine kinases, ZAP-70, and Syk. Recruitment of ZAP-70 or Syk to the phosphorylated ITAMs leads to the activation of these proteintyrosine kinases and subsequent tyrosine phosphorylation of multiple intracellular substrates.One substrate that is rapidly tyrosine-phosphorylated in response to TCR engagement is the 95-kDa protooncogene Vav1. Vav1 is expressed exclusively in hematopoietic cells. However, a homologous protein, Vav2, i...

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The pancreatic β-cell is critical for the maintenance of glycemic control. Knowing the compendium of genes expressed in β-cells will further our understanding of this critical cell type and may allow the identification of future antidiabetes drug targets. Here, we report the use of next-generation sequencing to obtain nearly 1 billion reads from the polyadenylated RNA of islets and purified β-cells from mice. These data reveal novel examples of β-cell-specific splicing events, promoter usage, and over 1000 long intergenic noncoding RNA expressed in mouse β-cells. Many of these long intergenic noncoding RNA are β-cell specific, and we hypothesize that this large set of novel RNA may play important roles in β-cell function. Our data demonstrate unique features of the β-cell transcriptome.

Given the importance of the Rho GTPase family member Rac1 and the Rac1/Cdc42 effector PAK1 in T-cell activation, we investigated the requirements for their activation by the T-cell receptor (TCR). Rac1 and PAK1 activation required the tyrosine kinases ZAP-70 and Syk, but not the cytoplasmic adaptor Slp-76. Surprisingly, PAK1 was activated in the absence of the transmembrane adaptor LAT while Rac1 was not. However, ef®cient PAK1 activation required its binding sites for Rho GTPases and for PIX, a guanine nucleotide exchange factor for Rho GTPases. The overexpression of bPIX that either cannot bind PAK1 or lacks GEF function blocked PAK1 activation. These data suggest that a PAK1±PIX complex is recruited to appropriate sites for activation and that PIX is required for Rho family GTPase activation upstream of PAK1. Furthermore, we detected a stable trimolecular complex of PAK1, PIX and the paxillin kinase linker p95PKL. Taken together, these data show that PAK1 contained in this trimolecular complex is activated by a novel LATand Slp-76-independent pathway following TCR stimulation.