Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling
Introduction
Members of the Sin1 family of proteins function in signal transduction and are essential for cellular stress responses and regulation of the actin cytoskeleton in several organisms. S. pombe Sin1 binds to the SAPK Sty1, and is required for the normal function of Sty1 [1]. The Dictyostelium Sin1 protein, RIP3, binds to activated Ras and has a critical role in chemotaxis and cAMP signal relay [2], [3]. Deletion of the gene encoding the S. cerevisiae Sin1 protein, Avo1p, resulted in defective polarization of the actin cytoskeleton [4]. Regulation of the actin cytoskeleton by Avo1 and RIP3 involves binding to the evolutionarily conserved TOR complex 2 (TORC2) [3], [4], [5], [6]. A single Sin1 gene is found in nearly all metazoan, yeast and amoeboid organisms, which together with a high level of evolutionary conservation suggests that Sin1 proteins may have a similar function in these organisms [7]. Human Sin1 (also known as MAPKAP1 and mSin1) mRNA is widely expressed in normal human tissues and can be alternatively spliced to encode distinct Sin1 protein isoforms [7]. Full-length Sin1 and a C-terminally truncated isoform, Sin1α, bind to JNK and can inhibit the activation of JNK by UV–C [8]. MIP1, a recently identified MEKK2-interacting protein that can suppress JNK signalling, is identical to the Sin1β isoform [9]. Ovine Sin1 mRNA can also be alternatively spliced, and full-length ovine Sin1 binds to the cytoplasmic tail of the type I interferon receptor [10].
The N-, K-, and H-Ras proto-oncogenes encode small GTPases that regulate cellular growth, differentiation and survival in response to extra-cellular stimuli such as growth factors and cellular stresses. Ras proteins are membrane-associated and cycle between an active GTP-bound state and an inactive GDP-bound state. Guanine nucleotide exchange factors (GEFs) recruited to the membrane activate Ras by catalysing the exchange of GDP for GTP. The activation of Ras is normally limited by GTPase-activating proteins (GAPs), which accelerate the intrinsic GTPase activity of Ras. However, mutations such as a Gly to Val substitution at residue 12 render Ras insensitive to the action of GAPs, and such constitutively activated Ras mutants are commonly found in human cancers [11], [12], [13], [14], [15]. The Ras proteins interact with and activate a diverse set of effector proteins, including c-Raf1, a serine/threonine kinase that regulates the ERK1/2 MAPK pathway, and phosphatidylinositol 3′-kinase (PI3K), a lipid kinase that regulates the Akt serine/threonine kinase [16], [17], [18]. Ras proteins can also activate the c-Jun N-terminal kinases (JNK) and the p38 MAPKs, however the pathways involved are not as clearly defined and may involve multiple effectors [19], [20].
Several observations suggest that a Ras-binding domain may be conserved in the orthologous Sin1 protein family. Dictyostelium RIP3 can bind to activated Ras and shows sequence similarity to the Raf-like Ras-binding domain (RBD) [2], [3]. S. cerevisiae Avo1 has been suggested to contain a Ras-association (RA) domain [4]. In addition, human Sin1 was first identified as a partial cDNA (JC310) that inhibited signalling by constitutively activated Ras in yeast [21]. We have investigated whether human Sin1 is involved in Ras signalling in mammalian cells, and herein report the identification of a Ras-binding domain (RBD) and a pleckstrin homology (PH) domain in Sin1. RBD and PH domains are present in nearly all Sin1 proteins, and are functionally active in human Sin1. Full-length Sin1 protein co-localised and interacted with Ras in vivo and its over-expression was found to inhibit Ras-mediated activation of ERK1/2, Akt and JNK signalling.
Section snippets
Similarity searches and secondary structure prediction
Full-length Sin1 sequences submitted to the GenBank non-redundant database were obtained as described elsewhere [7]. The Cryptococcus neoformans Sin1 sequence used here was a version of CNBJ2220 that was modified according to genomic sequence and homology to other Sin1 proteins. Similarity to hidden Markov models (HMMs) representing domains in the PFAM library was identified by BLAST (http://pfam.wustl.edu/-hmmsearch.shtml). The SMART domain library was searched at //smart.embl-heidelberg.de/
Sin1 family proteins contain a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain
To determine whether a potential Ras-binding domain has been conserved in the Sin1 protein family, the PFAM library of domains was searched using known Sin1 protein sequences as queries. These searches revealed that several lower eukaryotic Sin1 proteins contained low-level similarity to the RBD, including S. cerevisiae Avo1p (E = 0.45), Dictyostelium RIP3 (E = 0.74) and N. crassa EAA28559 (E = 0.25). Although the E-values for these alignments were not significant, the similarity occurred in regions
Discussion
Using sequence homology we show here that nearly all Sin1 orthologues contain potential Raf-like Ras-binding domains (RBDs) and pleckstrin homology (PH) domains. As these domains were not automatically predicted in searches of domain databases, this study demonstrates the power of analyzing orthologous sequences. Importantly, both the RBD and PH domains were shown to be functional in human Sin1. The human Sin1 RBD was shown to bind H-RasG12V, and Sin1 bound to and co-localised with activated
Acknowledgements
Wayne Schroder and Nicole Cloonan were supported by Australian Postgraduate Awards. The authors thank David James and Tobias Spielmann for providing reagents used in this study.
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