Schickwann Tsai

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558–724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90β and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61β subunit

We report here the isolation of a new member of the ADP‐ribosylation factor (ARF)‐like family (ARL‐6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage‐restricted expression, ARL‐6 mRNA increased with erythropoietin‐induced maturation of J2E cells, and decreased with interleukin 6‐induced differentiation of M1 monoblastoid cells. In tissues, ARL‐6 mRNA was most abundant in brain and kidney. While ARL‐6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP‐γS, like many members of the ARF/ARL family. Using the yeast two‐hybrid system, six molecules which interact with ARL‐6 were identified including SEC61β, a subunit of the heterotrimeric protein conducting channel SEC61p. Co‐immunoprecipitation of ARL‐6 confirmed a stable association between ARL‐6 and SEC61β in COS cells. These results demonstrate that ARL‐6, a novel member of the ADP‐ribosylation factor‐like family, interacts with the SEC61β subunit.

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T3) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T3 receptor, providing a link between the Epo and T3 signaling pathways. Trip-1 co-localized with Lyn and the T3 receptor α in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T3 stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T3 reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27Kip1 and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T3-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27Kip1, and blocking T3 response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

14-3-3zeta interacts with the alpha-chain of human interleukin 9 receptor.

Interleukin 9 (IL-9) exerts its pleiotropic effects through the IL-9 receptor (IL-9R) complex, which consists of the IL-9R alpha-chain, which determines the cytokine specificity, and the IL-2 receptor gamma-chain. In the present study we used a modified yeast two-hybrid system to isolate cDNA species encoding proteins that interacted with the intracellular domain of the human IL-9R alpha-chain (hIL-9Ralpha). We have identified 14-3-3zeta as an hIL-9Ralpha-interacting protein. We also mapped residues 518-522 (Arg-Ser(519)-Trp-Thr(521)-Phe) in hIL-9Ralpha and helix I of 14-3-3zeta as being important for interaction. Moreover, peptide competition experi-ments suggested that interaction between hIL-9Ralpha and 14-3-3zeta requires the phosphorylation of Ser(519) or Thr(521). This is the first demonstration that 14-3-3 can interact with a non-tyrosine kinase receptor. The interaction between 14-3-3 and IL-9Ralpha but not IL-4Ralpha also suggests a potential role for 14-3-3 in determining cytokine specificity.

Jagged2 induces cell cycling in confluent fibroblasts susceptible to density‐dependent inhibition of cell division

Jagged2 is a member of the DSL (Delta‐Serrate‐Lag‐2) ‐ligand family of transmembrane proteins that signal through the Notch receptors. In many cases of human acute lymphoblastic T‐cell leukemias, chromosomal translocations fuse a part of the Notch‐1 gene to the T‐cell receptor‐β locus (Ellison et al., 1991, Cell 66:649–661). The truncated Notch‐1 allele encodes an aberrant protein that lacks most of the extracellular domain and is constitutively activated (Pear et al., 1996, J Exp Med 183:2283–2291). A similarly truncated version of Notch‐1 was capable of transforming primary baby rat kidney cells in cooperation with the E1A oncogene of adenovirus (Capobianco et al., 1997, Mol Cell Bio 17:6265–6273). The transformed cells grew to a high population density in culture and were tumorigenic in vivo. It was unclear what roles Notch signaling played in neoplastic transformation. In this report, we demonstrate that sustained activation of the Jagged2/Notch signal transduction pathway induced continuous cell cycling in confluent rabbit‐skin fibroblasts sensitive to density‐dependent inhibition of cell division. The ability to overcome density‐dependent inhibition of cell division correlated with elevated cyclin‐dependent kinase‐2 (CDK2) activity and a lower level of induction of the CDK inhibitor p27 in the target cells. Similar cell‐cycle effect was seen when a truncated mouse Notch‐1 construct with constitutive activity was expressed. Taken together, our findings indicate that sustained activation of the Jagged2/Notch signal transduction pathway can overcome density‐dependent inhibition of cell division and therefore may contribute to neoplastic transformation. J. Cell. Physiol. 185:425–431, 2000.