Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4
Introduction
The suppressor of cytokine signalling (SOCS) proteins are a family of intracellular inhibitors of cytokine signal transduction (Endo et al., 1997, Naka et al., 1997, Starr et al., 1997, Yoshimura et al., 1995). Their expression is induced by cytokines in a range of tissues, and they appear to act in a classic negative feedback loop to regulate signal transduction (Bjorbaek et al., 1999, Starr et al., 1997). The eight known members of the SOCS family are characterized by a central SH2 domain, N-terminal regions of variable amino acid sequence, and a conserved carboxy-terminal motif termed the SOCS box (Hilton et al., 1998, Starr et al., 1997). Members of this group have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors, activities that require the amino-terminal and SH2 domains of the protein (Narazaki et al., 1998, Nicholson et al., 1999). Evidence suggests that the SOCS box serves to mediate interaction with the elongin B and C complex (Kamura et al., 1998, Zhang et al., 1999), possibly acting as an adaptor by which the SOCS proteins interact with the proteasomal degradation pathway. This proposed role for the SOCS box parallels that of the F-Box in the phospho-protein-ubiquitin ligase complex (PULC) that is utilized in the control of the cell cycle (Hershko and Ciechanover, 1998).
DNA database searches with a SOCS box consensus sequence revealed the existence of several other families of proteins containing a carboxy-terminal SOCS box that can be categorized according to the structural elements found upstream of this conserved domain (Hilton et al., 1998). As well as the members of the SOCS family proper, these include proteins containing WD40 repeats, SPRY domains, GTPase-like domains and ankyrin repeats.
We report here the cloning and characterization of members of the nkyrin repeat-containing OCS ox protein family, the Asbs. The ankyrin repeat is a loosely conserved sequence of approximately 33 amino acids that is found, in varying copy numbers, in proteins with a wide variety of functions (for a review, see Bork, 1993), including receptors (Wharton et al., 1985), cell-cycle regulators (Breeden and Nasmyth, 1987), membrane skeletal proteins (Lambert et al., 1990), secreted proteins (Grishin, 1998), tumour suppressors (Serrano et al., 1993), and transcription factors (LaMarco et al., 1991). From crystal structures and NMR analysis (Batchelor et al., 1998, Baumgartner et al., 1998, Gorina and Pavletich, 1996, Huxford et al., 1998, Mandiyan et al., 1999, Tevelev et al., 1996, Venkataramani et al., 1998, Yang et al., 1998) it is clear that ankyrin repeats are a generic structural motif involved in protein–protein interaction, with each repeat forming a V-shaped helix–turn–helix motif, and consecutive repeats stacking sequentially in bundles. In order to begin characterizing this novel protein family, we have cloned cDNAs and genomic sequence for Asb-1, Asb-2, Asb-3 and Asb-4, determined their chromosomal location, and examined their expression in adult mouse tissues.
Section snippets
Materials and methods
All restriction enzymes were obtained from Roche (Mannheim, Germany). Oligonucleotides were manufactured by Genset Pacific (Lismore, Australia) and Geneworks (Adelaide, Australia)
Asb cDNAs
Using radiolabelled oligonucleotides designed from EST sequence, two full-length Asb-1 cDNA clones were isolated from a mouse spleen library, sequencing of which revealed that Asb-1 is encoded by an open reading frame (ORF) of 1008 bp (GenBank Accession No. AF155352). The predicted protein of 336 amino acids comprises six ankyrin repeats and a C-terminal SOCS box, with an N-terminal region of 38 amino acids that contains no obvious amino acid sequence motifs (Fig. 1). 5′ RACE analysis suggested
Discussion
We have cloned full-length cDNAs encoding four members of the Asb family. Characterization of the 5′ end of the transcripts has confirmed that the predicted proteins have between six and 12 ankyrin repeats, short novel N-terminal regions, a C-terminal SOCS box, and, in the case of Asb-3, a short region C-terminal to the SOCS box. Sequencing of human ESTs shows that these novel proteins are highly conserved between mouse and human. The genes encoding the Asbs are organized into multiple exons,
Acknowledgements
This work was supported by the National Health and Medical Research Council, Canberra, the Anti-Cancer Council of Victoria, an Australian Government Cooperative Research Centres Program Grant, the National Institutes of Health, Bethesda, Grant No. CA22556, the J.D. and L. Harris Trust and AMRAD Operations Pty Ltd, Melbourne. B.T.K. is the recipient of an Australian Postgraduate Award.
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