The gene AML1/PEBP2␣B encodes the ␣ subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. We have identified a new isoform of the AML1 transcript, termed AML1⌬N, in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1⌬N transcript was detected in various hematopoietic cell lines of lymphoid to myeloid cell origin, as revealed by RNase protection and reverse transcriptase PCR analyses. The protein product of AML1⌬N lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the ␤ subunit. However, AML1⌬N was found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1⌬N in 32Dcl3 myeloid cells blocked granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1⌬N acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1.

APS (adapter protein with Pleckstrin homology and Src homology 2 domains) is recruited by the autophosphorylated insulin receptor and is essential for Glut4 translocation. Although both APS and CAP (c-CblInsulin increases glucose transport into target tissues by promoting the exocytosis of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (1, 2). In recent years, it has emerged that there are two pathways required for insulin-stimulated glucose transport, namely an insulin receptor substrate/PI 1 3-kinase pathway and a PI 3-kinase independent pathway involving the c-Cbl-associated protein (CAP) and the tyrosine phosphorylation of c-Cbl (2). Although c-Cbl was thought to be recruited by CAP, it has also emerged that APS (adapter protein with a PH and SH2 domain), a member of a larger adapter protein family, can also recruit c-Cbl following insulin-stimulation (3, 4). Following the binding of insulin, the insulin receptor undergoes autophosphorylation in the activation loop of the kinase domain. This provides docking sites for the binding of several proteins, including SH2-B and the APS adapter protein (5-8).The resulting recruitment of the APS adapter protein to the kinase domain allows it to undergo tyrosine phosphorylation on Tyr-618 (7). The phosphorylated Tyr-618 then allows APS to bind to the variant SH2 domain of c-Cbl (8). This is followed by the tyrosine phosphorylation of c-Cbl on tyrosines 700 and 774, resulting in phosphorylated c-Cbl binding to the SH2 domain of Crk (4, 9). It is also thought that the adapter protein CAP could facilitate interaction with the insulin receptor, because CAP was also identified as a c-Cbl binding protein using c-Cbl as a bait in a yeast two-hybrid screen and was shown to interact with the insulin receptor in intact cells (10). CAP contains an N-terminal region homologous to Sorbin (Sorbin homology/ SOHO domain) and three SH3 domains in the C terminus (10). CAP is constitutively bound to a proline-rich region in Cbl through its C-terminal SH3 domain (11). Recently, a number of new splice variants of CAP have been described (12). Following the insulin-stimulated phosphorylation of c-Cbl, the CAP/Cbl complex migrates to the caveolin-rich lipid rafts, a movement facilitated by the interaction of the CAP SOHO domain with flotillin, a protein in lipid rafts (13). This allows the Crk/C3G complex to be recruited to this microdomain, where C3G activates the small G protein TC10 (14). The activation of TC10 occurs independently of PI 3-kinase and is crucial for insulinstimulated Glut4 translocation (2).Since APS and CAP both interact with c-Cbl (3, 4, 8, 10), the relative individual importance and the temporal sequence of these proteins in recruiting c-Cbl has not been clear. In this study, we sought to determine whether both proteins were required to recruit c-Cbl to the insulin receptor or whether they were mutually exclusive. We addressed this question by coexpression of APS and CAP in cells that do not express these proteins and by silencing of th...

TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-Iike sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif.

The polyomavirus enhancer binding protein 2␣B (AML1͞PEBP2␣B͞Cbfa2) plays a pivotal role in granulocyte colony-stimulating factor (G-CSF)-mediated differentiation of a myeloid progenitor cell line, 32Dc13. In this article, we report the identification of a PEBP2␣B interacting protein, Ear-2, an orphan member of the nuclear hormone receptor superfamily that directly binds to and can inhibit the function of PEBP2␣B. Ear-2 is expressed in proliferating 32Dc13 cells in presence of interleukin 3 but is down-regulated during differentiation induced by G-CSF. Interestingly, AML1͞ ETO(MTG8), a leukemogenic chimeric protein can block the differentiation of 32Dc13 cells, which is accompanied by the sustained expression of ear-2. Overexpression of Ear-2 can prevent G-CSF-induced differentiation, strongly suggesting that ear-2 is a key negative regulator of granulocytic differentiation. Our results indicate that a dynamic balance existing between PEBP2␣B and Ear-2 appears to determine the choice between growth or differentiation for myeloid cells.

Nod-like receptors (NLRs) are cytosolic sensors for microbial molecules. Nucleotide-binding oligomerization domain (NOD)1 and NOD2 recognize the peptidoglycan derivatives, meso-diaminopimelic acid (meso-DAP) and muramyl dipeptide (MDP), respectively, and trigger host innate immune responses. In the present study, we examined the function of NOD1 and NOD2 on innate immune responses in human periodontal ligament (PDL) cells. The gene expression of NOD1 and NOD2 was examined by RT-PCR. IL-6 and IL-8 production in culture supernatants was measured by ELISA. Western blot analysis was performed to determine the activation of NF-κB and MAPK in response to Tri-DAP and MDP. The genes of NOD1 and NOD2 appeared to be expressed in PDL cells. Although the levels of NOD2 expression were weak in intact cells, MDP stimulation increased the gene expression of NOD2 in PDL cells. Tri-DAP and MDP led to the production of IL-6 and IL-8 and the activation of NF-κB and MAPK in PDL cells. Toll-like receptor (TLR) stimulation led to increased gene expression of NOD1 and NOD2 in PDL cells. Pam3CSK4 (a TLR2 agonist) and IFN-γ synergized with Tri-DAP and MDP to produce IL-8 and IL-6 in PDL cells. Our results indicate that NOD1 and NOD2 are functionally expressed in human PDL cells and can trigger innate immune responses.