Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.

Wnt binding to members of the seven-span transmembrane Frizzled (Fz) receptor family controls essential cell fate decisions and tissue polarity during development and in adulthood. The Fzmediated membrane recruitment of the cytoplasmic effector Dishevelled (Dvl) is a critical step in Wnt/β-catenin signaling initiation, but how Fz and Dvl act together to drive downstream signaling events remains largely undefined. Here, we use an Fz peptide-based microarray to uncover a mechanistically important role of the bipartite Dvl DEP domain and C terminal region (DEP-C) in binding a three-segmented discontinuous motif in Fz. We show that cooperative use of two conserved motifs in the third intracellular loop and the classic C-terminal motif of Fz is required for DEP-C binding and Wnt-induced β-catenin activation in cultured cells and Xenopus embryos. Within the complex, the Dvl DEP domain mainly binds the Fz C-terminal tail, whereas a short region at the Dvl C-terminal end is required to bind the Fz third loop and stabilize the Fz-Dvl interaction. We conclude that Dvl DEP-C binding to Fz is a key event in Wnt-mediated signaling relay to β-catenin. The discontinuous nature of the Fz-Dvl interface may allow for precise regulation of the interaction in the control of Wnt-dependent cellular responses.peptide microarray | protein-protein interaction | Wingless signaling

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The NF-B signaling pathway plays a crucial role in the immune, inflammatory, and apoptotic responses. Recently, we identified the NF-B Essential Modulator (NEMO) as an essential component of this pathway. NEMO is a structural and regulatory subunit of the high molecular kinase complex (IKK) responsible for the phosphorylation of NF-B inhibitors. Data base searching led to the isolation of a cDNA encoding a protein we called NRP (NEMO-related protein), which shows a strong homology to NEMO. Here we show that NRP is present in a novel high molecular weight complex, that contains none of the known members of the IKK complex. Consistently, we could not observe any effect of NRP on NF-B signaling. Nonetheless, we could demonstrate that treatment with phorbol esters induces NRP phosphorylation and decreases its half-life. This phosphorylation event could only be inhibited by K-252a and stauroporin. We also show that de novo expression of NRP can be induced by interferon and tumor necrosis factor ␣ and that these two stimuli have a synergistic effect on NRP expression. In addition, we observed that endogenous NRP is associated with the Golgi apparatus. Analogous to NEMO, we find that NRP is associated in a complex with two kinases, suggesting that NRP could play a similar role in another signaling pathway.The transcription factor NF-B plays a pivotal role in many cellular processes such as immune responses, inflammation, and apoptosis (1, 2). NF-B is composed of homo-and heterodimers of various members of the NF-B/Rel family (3, 4) and is retained in an inactive form in the cytoplasm by an inhibitory protein belonging to the IB family, mainly represented by IB␣, IB␤, and IB⑀ (5-7).In response to diverse stimuli, including IL-1, 1 LPS, TNF␣, or PMA, as well as several viral proteins, active NF-B translocates to the nucleus as a result of the complete proteolytic degradation of the IB proteins. This mechanism has been best studied for the inhibitor IB␣ and demonstrated to involve phosphorylation on two specific serine residues (8 -13) followed by polyubiquitination and degradation by the 26 S proteasome (14). More recently a specific serine protein kinase activity responsible for IB␣ phosphorylation has been identified as a large cytoplasmic complex (600 -800 kDa) containing two catalytic subunits (IKK1/␣ and IKK2/␤) (15-19). IKK␣ and IKK␤ are related molecules of 85 and 87 kDa, respectively, and share 50% sequence similarity. Both proteins contain NH 2 -terminal kinase domains, leucine zipper, and helix-loop-helix motifs (16,19). In vitro phosphorylation studies have shown that both kinases can phosphorylate IB␣ on serines 32 and 36, but IKK␤ is more active in this regard.Recently, we have cloned, by complementation of an NF-B unresponsive cell line, a third component of the IKK complex (20), that we called NEMO (NF-B Essential Modulator). NEMO is a 48-kDa glutamine-rich protein, which lacks a catalytic subunit, but contains two coiled-coil motifs, a leucine zipper, and a COOH-terminal zinc finger (20). NEMO is a regula...