The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-B (NF-B) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-B in both in vivo and in vitro proteinprotein interaction assays. FBI-1 enhanced NF-B-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IB␣ or IB␤. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-B-mediated transcription, and the repression was cooperative with IB␣ or IB␤. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-B-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-B movement into the nucleus and increased the stability of NF-B in the nucleus, which enhanced NF-B-mediated transcription of the E-selectin gene. FBI-1 also interacted with IB␣ and IB␤.The BTB/POZ (broad complex, Tramtrack, and bric-a-brac/ poxvirus and zinc finger) domain is an evolutionarily conserved protein-protein interaction domain that is found at the N terminus of various cellular and viral regulatory proteins. The proteins containing the BTB/POZ domain have several C-terminal structures important in their biological functions, such as the zinc finger, actin-binding repeats, and ion channel motifs (1-3). The POZ domains of PLZF (promyelocytic leukemia zinc finger) and Bcl-6 (B-cell lymphoma-6) have been shown to interact with SMRT (silencing mediator for retinoid and thyroid hormone receptors)/N-CoR (nuclear receptor co-repressor), mSin3A, and histone deacetylases (4, 5).FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) was purified as a cellular factor that binds specifically to the wild-type IST (inducer of short transcripts) elements of human immunodeficiency virus-1 long terminal repeats and the proximal promoter of the ADH5/FDH gene, and its cDNA was cloned (6 -9). FBI-1 is a ubiquitous transcription factor that contains a BTB/POZ domain at its N terminus and Krü ppel-like zinc fingers at its C terminus. There have been several recent reports on the function of FBI-1. FBI-1 stimulates Tat (transactivator of transcription) activity on the human immunodeficiency virus-1 long terminal repeat (8) and represses human ADH5/FDH gene expression by interacting with Sp1 zinc fingers (9). The mouse counterpart of FBI-1, LRF (leukemia/lymphoma-related factor), is co-immunoprecipitated and co-localized with Bcl-6 (...

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C 2 H 2 -type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GCbox recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.Transcriptional regulation of the eukaryotic gene is a complicated process, involving a series of complex molecular interactions among regulatory and transcription factors. Specificity protein 1 (Sp1) 1 is probably one of the best characterized sequence-specific transcription factors, and has numerous functions in the transcription of many cellular and viral genes harboring GC boxes in their promoters (1, 2). Sp1 and its family of proteins have been implicated in a host of essential biological processes, and have been proven important in apoptosis, cell growth inhibition, differentiation, and carcinogenesis (Refs. 3-5 and references therein).Sp1 is a member of the Sp-multigene family, which also includes Sp2, Sp3, Sp4, Sp5, Sp6, Sp7, and Sp8 (3-5). The Sp-family proteins exhibit similar domain structures and are evolutionarily closely related. All of these proteins possess highly conserved C 2 H 2 -type zinc finger DNA binding domains at their C termini, and all belong to the Krü ppel-like zinc finger superfamily (3-5). In addition, the Sp-family proteins have been demonstrated to undergo post-translational modifications as the result of diverse mechanisms. For example, Sp1 is glycosylated and phosphorylated by Erk2, protein kinase C, casein kinase II, and cAMP-dependent protein kinase; Sp3 is acetylated and SUMOylated by the protein inhibitor of activated STAT (PIAS1, Ref. 3 and references therein, Refs. 6 -10).The Sp1 protein co...

Dual specific protein kinase Dyrks are thought to play a key role in the regulation of cell growth in a variety of cellular systems. Interestingly, human Dyrk1 is mapped to the Down's syndrome (DS) critical region on chromosome 21, and thought to be a candidate gene responsible for the mental retardation of DS patients. Huntingtin-interacting protein 1 (Hip-1), a proapoptotic mediator, is implicated as a molecular accomplice in the pathogenesis of Huntington's disease. In the present study we found that Dyrk1 selectively binds to and phosphorylates Hip-1 during the neuronal differentiation of embryonic hippocampal neuroprogenitor (H19-7) cells. The Dyrk1-mediated phosphorylation of Hip-1, in response to bFGF, resulted in the blockade of Hip-1-mediated neuronal cell death as well as the enhancement of neurite outgrowth. Furthermore, the addition of etoposide to proliferating H19-7 cells caused the diminished binding of Hip-1 to Dyrk1 and the levels of phosphorylated Hip-1 remarkably decreased. Simultaneously, the dissociated Hip-1 from Dyrk1 bound to caspase-3 in response to etoposide, which led to its activation and consequently cell death in H19-7 cells. These data suggest that the phosphorylation of Hip-1 by Dyrk1 has a dual role in regulating neuronal differentiation and cell death. The interaction between Dyrk1 and Hip-1 appeared to be differentially modulated by different kinds of stimuli, such as bFGF and etoposide in H19-7 cells.

Tat activates transcription by interacting with Sp1, NF-B, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-yinhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.