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.     

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。     

The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites

We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.     

The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.     

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.     

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.     

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.     

Sam68 relocalization into stress granules in response to oxidative stress through complexing with TIA-1

Sam68 has been implicated in a variety of important cellular processes such as RNA metabolism and intracellular signaling. We have recently shown that Sam68 cytoplasmic mutants induce stress granules (SG) and inhibit HIV-1 nef mRNA translation [J. Henao-Mejia, Y. Liu, I.W. Park, J. Zhang, J. Sanford, J.J. He, Suppression of HIV-1 Nef translation by Sam68 mutant-induced stress granules and nef mRNA sequestration, Mol. Cell 33 (2009) 87-96]. These findings prompted us to investigate the possibility and the underlying mechanisms of the wild-type counterpart Sam68 SG recruitment. Herein, we revealed that Sam68 was significantly recruited into cytoplasmic SG under oxidative stress. We then demonstrated that domain aa269-321 and KH domain were both essential for this recruitment. Nevertheless, Sam68 knockdown had no effects on SG assembly, indicating that Sam68 is not a constitutive component of the SG. Moreover, we showed that Sam68 cytoplasmic mutant-induced SG formation was independent of eIF2α phosphorylation. Lastly, we demonstrated that Sam68 was complexed with T-cell intracellular antigen-1 (TIA-1), a core SG component, and that the complex formation was correlated with Sam68 SG recruitment. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1 in response to oxidative stress and suggest that cytoplasmic SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.     

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.     

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.     

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.     

Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.Key words: PTK6, BRK, Sik, SAM68, prostate cancer, nuclear localization, tyrosine kinase     

Fyn membrane localization is necessary to induce the constitutive tyrosine phosphorylation of Sam68 in the nucleus of T lymphocytes.

A close relationship between Sam68, a tyrosine and proline-rich RNA-binding protein, and Src protein tyrosine kinases (PTK) has already been established, also in T lymphocytes. A constitutive phosphorylation of the molecule has also been documented in various transformed T cells, which probably reflects an increased expression of PTK of the Src family. Using the hybridoma T cell line, T8.1, or Jurkat T cells, we investigated the respective contribution of the two Src kinases Fyn and Lck, expressed in T cells, in this phenomenon. By overexpressing the two proteins, we show that the constitutive phosphorylation of Sam68 in vivo directly correlates with cellular Fyn levels, but not with Lck expression, despite the capacity of the PTK to strongly phosphorylate the molecule in vitro. Overexpressed Fyn is mainly localized at the cell membrane. We find that Sam68 phosphorylation, including in the nuclear fraction in which the molecule is predominantly expressed, is lost with a delocalized Fyn mutant deleted of its N-terminal membrane-anchoring domain. Finally, we demonstrate, using a construct encoding a Sam68 molecule without its nuclear localization signal, that nuclear expression of Sam68 is not required for phosphorylation. We conclude that the constitutive phosphorylation of Sam68 in T cells is a Fyn-dependent process occurring in a cell-membrane compartment from which phospho-Sam68 molecules can thereafter accumulate into the nucleus.