Specificity of Rel-inhibitor interactions in Drosophila embryos

The Rel family of transcription factors participate in a diverse array of processes, including acute responses to injury and infection, lymphocyte differentiation, and embryonic patterning. These proteins show homology in an extended region spanning about 300 amino acids (the Rel homology domain [RHD]). The RHD mediates both DNA binding and interactions with a family of inhibitor proteins, including I kappa B alpha and cactus. Previous studies have shown that an N-terminal region of the RHD (containing the sequence motif RXXRXRXXC) is important for DNA binding, while the C-terminal nuclear localization sequence is important for inhibitor interactions. Here we present a structure-function analysis of the Drosophila dorsal RHD. These studies identify another sequence within the RHD (region I) that is essential for inhibitor interactions. There is a tight correlation between the conservation of region I sequences and the specificity of Rel-inhibitor interactions in both flies and mammals. Point mutations in the region I sequence can uncouple DNA binding and inhibitor interactions in vitro. The phenotypes associated with the expression of a modified dorsal protein in transgenic Drosophila embryos suggest a similar uncoupling in vivo. Recent crystallographic studies suggest that the region I sequence and the nuclear localization sequence might form a composite surface which interacts with inhibitor proteins.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  P. Sigler,et al.  Structure of NF-κB p50 homodimer bound to a κB site , 1998, Nature.

[3]  S. Wasserman,et al.  Dorsal, a Drosophila Rel-like protein, is phosphorylated upon activation of the transmembrane protein Toll , 1994, Molecular and cellular biology.

[4]  M. Levine,et al.  Regulation of the dorsal morphogen by the Toll and torso signaling pathways: a receptor tyrosine kinase selectively masks transcriptional repression. , 1994, Genes & development.

[5]  K. Anderson,et al.  The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo , 1994, Cell.

[6]  D. Hultmark.  Ancient relationships , 1994, Nature.

[7]  M. Levine,et al.  Dif, a dorsal-related gene that mediates an immune response in Drosophila , 1993, Cell.

[8]  R. Steward,et al.  Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal , 1993, The Journal of cell biology.

[9]  A. Baldwin,et al.  The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. , 1993, Genes & development.

[10]  C. Gélinas,et al.  I kappa B alpha-mediated inhibition of v-Rel DNA binding requires direct interaction with the RXXRXRXXC Rel/kappa B DNA-binding motif. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Meister,et al.  Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila. , 1993, Comptes rendus de l'Academie des sciences. Serie III, Sciences de la vie.

[12]  R. Hay,et al.  Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhinition of NF-ϰB binding activity , 1993 .

[13]  M. Levine,et al.  Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila. , 1993, Genes & development.

[14]  S. Wasserman.  A conserved signal transduction pathway regulating the activity of the rel-like proteins dorsal and NF-kappa B. , 1993, Molecular biology of the cell.

[15]  C. Rushlow,et al.  Conversion of a silencer into an enhancer: evidence for a co‐repressor in dorsal‐mediated repression in Drosophila. , 1993, The EMBO journal.

[16]  M. Levine,et al.  Conversion of a dorsal‐dependent silencer into an enhancer: evidence for dorsal corepressors. , 1993, The EMBO journal.

[17]  David Baltimore,et al.  Regulation of the NF-?B/rel transcription factor and I?B inhibitor system , 1993 .

[18]  A. Baldwin,et al.  Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation , 1993, Molecular and cellular biology.

[19]  W C Greene,et al.  NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. , 1993, Science.

[20]  S. Wasserman,et al.  pelle encodes a protein kinase required to establish dorsoventral polarity in the Drosophila embryo , 1993, Cell.

[21]  I. Orlow,et al.  Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by IL-1 beta. , 1993, Science.

[22]  U. Zabel,et al.  Nuclear uptake control of NF‐kappa B by MAD‐3, an I kappa B protein present in the nucleus. , 1993, The EMBO journal.

[23]  S. Kidd.  Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins , 1992, Cell.

[24]  C. Nüsslein-Volhard,et al.  cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the IκB gene family of vertebrates , 1992, Cell.

[25]  M. Levine,et al.  The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. , 1992, Genes & development.

[26]  M. Levine,et al.  Individual dorsal morphogen binding sites mediate activation and repression in the Drosophila embryo. , 1992, The EMBO journal.

[27]  J. Casanova.  Interaction between torso and dorsal, two elements of different transduction pathways in the Drosophila embryo , 1991, Mechanisms of Development.

[28]  D Kosman,et al.  The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos. , 1991, Genes & development.

[29]  J. Foldi,et al.  Constitutive and NF-kappa B-like proteins in the regulation of the serum amyloid A gene by interleukin 1. , 1991, Cytokine.

[30]  K. Anderson,et al.  Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo. , 1991, Genes & development.

[31]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[32]  M. Levine,et al.  The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in drosophila , 1991, Cell.

[33]  M. Levine,et al.  The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila , 1989, Cell.

[34]  R. Steward.  Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function , 1989, Cell.

[35]  C. Nüsslein-Volhard,et al.  A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo , 1989, Cell.

[36]  K. Anderson,et al.  The role of easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo , 1989, Cell.

[37]  C. Thummel,et al.  Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. , 1988, Gene.

[38]  K. Anderson,et al.  The Toll gene of drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein , 1988, Cell.

[39]  U. Sieben,et al.  STRUCTURE, REGULATION AND FUNCTION OF NF-1d3 , 1994 .

[40]  G. Franzoso,et al.  Structure, regulation and function of NF-kappa B. , 1994, Annual review of cell biology.

[41]  R. Hay,et al.  Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity. , 1993, Nucleic Acids Research.

[42]  R. Steward,et al.  Homeostatic balance between dorsal and cactus proteins in the Drosophila embryo. , 1993, Development.

[43]  M. Frasch,et al.  Maternal regulation of zerknüllt: a homoeobox gene controlling differentiation of dorsal tissues in Drosophila , 1987, Nature.