Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.     

Transmembrane neuregulins interact with LIM kinase 1, a cytoplasmic protein kinase implicated in development of visuospatial cognition

The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of LIM domain interactions with receptor tyrosine kinases

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.     

Structure of the murine CD156 gene, characterization of its promoter, and chromosomal location

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.