Met receptors induce Sam68-dependent cell migration by activation of alternate extracellular signal-regulated kinase family members

The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression, and invasive cancer growth. Similarly, altered expression and splicing (i.e. inclusion of variant exon 5, "v5") of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to further understand how HGF regulates CD44v5 expression. Immortalized nontumorigenic keratinocyte (HaCaT) cells abundantly express both Met receptors and CD44v5 transmembrane glycoproteins. HGF stimulated CD44v5 protein expression and HaCaT cell migration; these events required activation of the ERK1/2 MAPK module and Sam68, a protein involved in RNA processing, splicing, and v5 inclusion. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, MDA-MB-231 cell migration occurred independently of ERK1/2 and CD44v5 expression and instead required ERK5 signaling to Sam68. Phospho-mutant, but not WT-Sam68, blocked HGF-induced cell migration in both cell types; MDA-MB-435 cells behaved similarly. These results suggest that Sam68 acts as a convergence point for ERK signaling to cell migration; blockade of phospho-Sam68 may provide a new avenue for therapeutic inhibition of metastatic cancers.     

Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.

Alternative splicing of pre-mRNA is a fundamental mechanism of differential gene expression in that it can give rise to functionally distinct proteins from a single gene, according to the developmental or physiological state of cells in multicellular organisms. In the pre-mRNA of the cell surface molecule CD44, the inclusion of up to 10 variant exons (v1-v10) is regulated during development, upon activation of lymphocytes and dendritic cells, and during tumour progression. Using minigene constructs containing CD44 exon v5, we have discovered exonic RNA elements that couple signal transduction to alternative splicing. They form a composite splice regulator encompassing an exon recognition element and splice silencer elements. Both type of elements are necessary to govern cell type-specific inclusion of the exon as well as inducible inclusion in T cells after stimulation by concanavalin A, by Ras signalling or after activation of protein kinase C by phorbol ester. Inducible splicing does not depend on de novo protein synthesis. The coupling of signal transduction to alternative splicing by such elements probably represents the mechanism whereby splice patterns of genes are established during development and can be changed under physiological and pathological conditions.     

Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion

The multiple isoforms of the transmembrane glycoprotein CD44 are produced by alternative RNA splicing. Expression of CD44 isoforms containing variable 5 exon (v5) correlates with enhanced malignancy and invasiveness of some tumors. Here we demonstrate that SRm160, a splicing coactivator, regulates CD44 alternative splicing in a Ras-dependent manner. Overexpression of SRm160 stimulates inclusion of CD44 v5 when Ras is activated. Conversely, small interfering RNA (siRNA)-mediated silencing of SRm160 significantly reduces v5 inclusion. Immunoprecipitation shows association of SRm160 with Sam68, a protein that also stimulates v5 inclusion in a Ras-dependent manner, suggesting that these two proteins interact to regulate CD44 splicing. Importantly, siRNA-mediated depletion of CD44 v5 decreases tumor cell invasion. Reduction of SRm160 by siRNA transfection downregulates the endogenous levels of CD44 isoforms, including v5, and correlates with a decrease in tumor cell invasiveness.     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。     

Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain

Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel α-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68.     

Identification of a Sam68 Ribonucleoprotein Complex Regulated by Epidermal Growth Factor

Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of ∼40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200–450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.     

Comprehensive analysis of interactions between the Src-associated protein in mitosis of 68 kDa and the human Src-homology 3 proteome

The protein Sam68 is involved in many cellular processes such as cell-cycle regulation, RNA metabolism, or signal transduction. Sam68 comprises a central RNA-binding domain flanked by unstructured tails containing docking sites for signalling proteins including seven proline-rich sequences (denoted P0 to P6) as potential SH3-domain binding motifs. To comprehensively assess Sam68-SH3-interactions, we applied a phage-display screening of a library containing all approx. 300 human SH3 domains. Thereby we identified five new (from intersectin 2, the osteoclast stimulating factor OSF, nephrocystin, sorting nexin 9, and CIN85) and seven already known high-confidence Sam68-ligands (mainly from the Src-kinase family), as well as several lower-affinity binders. Interaction of the high-affinity Sam68-binders was confirmed in independent assays in vitro (phage-ELISA, GST-pull-down) and in vivo (FACS-based FRET-analysis with CFP- and YFP-tagged proteins). Fine-mapping analyses with peptides established P0, P3, P4, and P5 as exclusive docking-sites for SH3 domains, which showed varying preferences for these motifs. Mutational analyses identified individual residues within the proline-rich motifs being crucial for the interactions. Based on these data, we generated a Sam68-mutant incapable of interacting with SH3 domains any more, as subsequently demonstrated by FRET-analyses. In conclusion, we present a thorough characterization of Sam68's interplay with the SH3 proteome. The observed interaction between Sam68 and OSF complements the known Sam68-Src and OSF-Src interactions. Thus, we propose, that Sam68 functions as a classical scaffold protein in this context, assembling components of an osteoclast-specific signalling pathway.     

T-STAR的结构及生物学功能

T-STAR基因定位于染色体8q24.2,其表达产物为分子量约55kDa的Sam68样蛋白,是STAR(signaltransductionandactivatorofRNA)家族新成员,具有RNA结合蛋白特征性的结合位点和酪氨酸磷酸化功能域。可能通过酪氨酸激酶信号转导系统和pre-mRNA的选择性剪接、加工等途径,参与了精子的发生、细胞的增殖调控、转化细胞的永生化过程并可能与某些疾病有关。     

Sam68 regulates a set of alternatively spliced exons during neurogenesis

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.     

Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated.     

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.     

The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4.

Exon enhancers are accessory pre-mRNA splicing signals that stimulate exon splicing. One class of proteins, the serine-arginine-rich (SR) proteins, have been demonstrated to bind enhancers and activate splicing. Here we report that A/C-rich exon enhancers (ACE elements) are recognized by the human YB-1 protein, a non-SR protein. Sequence-specific binding of YB-1 was observed both to an ACE derived from an in vivo iterative selection protocol and to ACE elements in an alternative exon (v4) from the human CD44 gene. The ACE element that was the predominant YB-1 binding site in CD44 exon v4 was required for maximal in vivo splicing and in vitro spliceosome assembly. Expression of wild-type YB-1 increased inclusion of exon v4, whereas a truncated form of YB-1 did not. Stimulation of exon v4 inclusion by wild-type YB-1 required the ACE necessary for YB-1 binding in vitro, suggesting that YB-1 stimulated exon inclusion in vivo by binding to an exonic ACE element. These observations identify a protein in addition to SR proteins that participates in the recognition of exon enhancers.     

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.