Summary Nek6 and Nercc1/Nek9 belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase/metaphase arrest, and depletion of XNercc from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5, a kinesin necessary for spindle bipolarity. Nek6 phosphorylates Eg5 at several sites in vitro, and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. While Cdk1 phosphorylates nearly all Eg5 during mitosis at Thr926, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion arrests cells with a monopolar spindle; this can be rescued by Eg5 wildtype but not by Eg5(Thr926Ala). Eg5(Ser1033Ala) rescues half as well as wildtype whereas an Eg5(Ser1033Asp) mutant is nearly as effective. Thus Nek6 phosphorylates a subset of Eg5 polypeptides during mitosis at a conserved site, whose phosphorylation is critical for the mitotic function of Eg5.

The 5-flanking region of the human Sp1 gene was cloned and characterized. Sequence analysis of this region showed the absence of both CAAT and TATA boxes and an initiator element. The proximal promoter of the Sp1 gene is a GC-rich region that contains multiple GC boxes and Ap2 binding sites. The major transcription start site is located 63 base pairs upstream of the translation start site. Transfection experiments demonstrate that all the elements necessary to achieve significant basal transcription activity are located between positions ؊443 and ؊20 relative to the translational start. Sp1 and Sp3 proteins bind to the downstream GC box located in the proximal promoter of Sp1. Furthermore, we demonstrate that the Sp1 protein activates Sp1 transcription activity; thus the Sp1 gene is autoregulated.Transcription factor Sp1 was originally identified by its binding to the multiple GC boxes in the simian virus 40 (SV40) early promoter (1, 2, 3) and the thymidine kinase promoter (4). Sp1 belongs to a small protein family, which is presently composed of Sp2, Sp3, and Sp4. This family contains a highly conserved DNA-binding domain composed of three zinc fingers close to the C terminus and serine-, threonine-, and glutaminerich domains in their N-terminal regions. The 81-amino acid C2H2-type zinc finger region, which comprises the DNA-binding domain, is the most highly conserved part of the protein.The two glutamine-rich regions (termed A and B) can act as strong activation domains (5, 6). Synergistic activation of promoters by Sp1 through multiple GC boxes also requires a short C-terminal domain (termed D) (7). More recently, an inhibitory domain has been mapped to the N terminus (8). Sp1 can be phosphorylated, a modification that affects its binding to the DNA (9 -11) and O-glycosylated (12), which confers resistance to proteosome-dependent degradation (13).Sp1 is able to form homotypic interactions producing multimeric complexes (14) as well as many heterotypic interactions. Sp1 associates directly with members of the basal transcription machinery, including the TATA box binding protein (15), the TATA box binding protein-associated factors dTAFII110/hTAFII130 (16), and hTAFII55 (17). Furthermore, Sp1 physically interacts and functionally cooperates with several transcription activators including OTF-1 (18), Oct-1 (19), transcription factor YY1 (identical to hTAFII55) (20,21), and the CCAAT-binding transcription factor NF-YA (22). In addition, Sp1 can interact with cell cycle regulators like p107 (23), E2F (24), and the retinoblastoma protein (25). In the latter case the protein association increases Sp1 transcriptional activity.The GC-rich boxes bound by Sp1 are also recognized by Sp3. Thus, these two Sp family members compete for DNA binding, and the resulting Sp transcriptional activity depends on a given ratio of Sp1/Sp3. This ratio varies by cell type and is subject to changes in the cell cycle or cellular conditions (26,27). It is also not clear whether Sp3 acts as an activator or as a repressor of Sp1-mediated t...

.es A.Bruna and M.Nicola Ás contributed equally to this workInhibition of the c-Jun N-terminal kinase (JNK) pathway by glucocorticoids (GCs) results in AP-1 repression. GC antagonism of AP-1 relies mainly on the transrepression function of the GC receptor (GR) and mediates essential physiological and pharmacological actions. Here we show that GCs induce the disassembly of JNK from mitogen-activated protein kinase kinase 7 (MKK7) by promoting its association with GR. Moreover, we have characterized a hormoneregulated JNK docking site in the GR ligand-binding domain that mediates GR±JNK interaction. The binding of GR to JNK is required for inhibition of JNK activation and induction of inactive JNK nuclear transfer by GCs. The dissociation of these two hormone actions shows that JNK nuclear transfer is dispensable for the downregulation of JNK activation by GCs. Nonetheless, nuclear accumulation of inactive JNK may still be relevant for enhancing the repression of AP-1 activity by GCs. In this regard, chromatin immunoprecipitation assays show that GC-induced GR±JNK association correlates with an increase in the loading of inactive JNK on the AP-1-bound response elements of the c-jun gene. Keywords: AP-1 antagonism/cell signaling/cross-talk/ MAPK docking site/MAPK pathway Introduction Glucocorticoids (GCs) play key physiological roles in development, cellular proliferation and differentiation. In addition, the prominent pharmacological actions of these hormones have prompted their widespread medical use to treat diverse pathological conditions such as asthma, allergic rhinitis, rheumatoid arthritis and leukemia (Barnes, 1998). GCs exert most of their actions by binding to an intracellular GC receptor (GR), a ligand-activated transcriptional regulator that belongs to the nuclear receptor (NR) superfamily (Beato et al., 1995).In most circumstances, hormone-free GR is associated with heterotypic complexes that contain chaperones, such as Hsp90, and co-chaperones, and is retained in the cytoplasm. Upon ligand binding, the chaperone complex is released and hormone-bound GR is rapidly transferred into the nucleus. Hormone-activated GR regulates gene transcription, either positively or negatively, by two major modes of action. The most well known involves the binding of GR homodimers to the GC response elements (GREs) found in the regulatory sequences of GC target genes. A second and more elusive mode of action is independent of the direct interaction of GR with DNA and relies on the interference (thus, also known as cross-talk or transrepression) with the activity of other transcriptional regulators by mechanisms based on protein±protein interactions. In contrast to the former, transrepression is apparently mediated by GR monomers. In fact, transactivation-defective mutants of GR, which cannot dimerize (GR dim ) or bind DNA (GRLS7), are fully competent in transrepression (Heck et al., 1994;Helmberg et al., 1995). Remarkably, the in vivo relevance of the DNA bindingindependent actions of GR has been evidenced by the genera...

We analysed in detail the minimal promoter of transcription factor Sp1, which extends 217 bp from the initiation of transcription. Within this sequence we identified putative binding sites for Sp1, nuclear factor Y (NF-Y), activator protein 2 ('AP-2'), CCAAT/enhancer-binding protein ('C/EBP') and E2F transcription factors. In one case, the boxes for Sp1 and NF-Y are overlapping. Gel-shift and supershift assays demonstrated specific binding of Sp1, Sp3 and NF-Y proteins. Transient transfections and luciferase assays revealed activation of the Sp1 minimal promoter upon overexpression of Sp1 itself, NF-Y and E2F. Whereas overexpression of NF-Y or E2F had an additive effect on Sp1 overexpression, the activation of Sp1 transcription due to Sp1 was counteracted by Sp3 overexpression. Mutagenesis analysis of the NFY/Sp1-overlapping box revealed that both factors compete for this box, and that when the NF-Y site of this overlapping box is specifically mutated there is an increase in Sp1 binding, thus increasing transcriptional activity. These results help to explain the complex regulation of the Sp1 gene, which depends on the relative amounts of Sp1, Sp3, E2F and NF-Y proteins in the cell.

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains &-acting elements for the transcription factors Spl and E2F. Given the ability of Spl to activate the dhfr promoter, we have evaluated the contribution of Spl to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Spl to the dhfr promoter is cell-growthphase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Spl by immunofluorescence reveals a decrease of this protein in the process leading to the GO state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Spl progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Spl in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Spl-binding or E2F-binding sites in the dhfr minimal promoter.Keywords: Spl, dihydrofolate reductase; proliferation; cell-cycle; transcription.Dihydrofolate reductase (DHFR) catalyzes the synthesis of purines, thymidylate and glycine, and is therefore needed for the replication of DNA in the S phase of the cell cycle. Although transcription of the dhfr gene increases dramatically at the G1/ S boundary [I-31, dhfr mRNA is present throughout the cell cycle [ 1, 41, accounting for a steadily increasing DHFR enzymic activity [5]. Considerable efforts have been made to identify the constitutive factors that control the dhfr promoter, and the factors that confer cell-cycle regulation on this gene. In this regard, the dhfr promoter contains cis-acting elements for the transcription factors Spl and E2F. It has been demonstrated that distinct GC boxes, responsible for Spl binding, direct transcription at two principal transcriptional initiation sites in rodents [6, 71. Only the most proximal GC box, at least in hamster cells, is needed to correctly initiate transcription at the major proximal site (minimum promoter). Deletion of this GC box abolishes transcription of the dhfr gene [8], despite the presence of the E2F-binding element just downstream of the major transcriptional start. It has also been shown that E2F from human cells (HeLa or HEL), either in nuclear extracts or purified, is able to bind to its recognition sequence in the murine [9] and hamster [lo, 111 dhfr genes, and that the E2F site is required for growth regulation 112, 131. Given that E2F can be bound by the tumor suppressor and cell-cycle regulator retinoblastoma protein (Rb) [14-171 these findings support a model for dhfr gene expression in which hypophosphorylated Rb represses E2F in resting cells, t...