This gene encodes a member of the K homology domain-containing, RNA-binding, signal transduction-associated protein family. The encoded protein appears to have many functions and may be involved in a variety of cellular processes, including alternative splicing, cell cycle regulation, RNA 3'-end formation, tumorigenesis, and regulation of human immunodeficiency virus gene expression.
Sam68 (the Src-Associated substrate in Mitosis of 68 kDa) is officially called KHDRBS1 (KH domain containing, RNA binding, signal transduction associated 1). Sam68 is a KH-type RNA binding protein that recognizes U(U/A)AA direct repeats with relative high affinity. Sam68 is predominantly nuclear and its major function in the nucleus is to regulate alternative splicing by recognizing RNA sequences neighboring the included/excluded exon(s). Direct evidence for the involvement of Sam68 in alternative splicing has been shown in promoting the inclusion of the variable exon 5 (v5) in CD44 correlating with cell migration potential. In addition, Sam68 in conjunction with hnRNPA1 influences the choice of the alternative 5' splice sites of Bcl-x regulating pro-survival and apoptotic pathways. The role of Sam68 was further highlighted in spinal muscular atrophy (SMA), as Sam68 promotes the skipping of exon 7 leading to a non-functional SMN2 protein. Sam68 was demonstrated to be involved in the alternative splicing of mRNAs implicated in normal neurogenesis using splicing-sensitive microarrays. Sam68 was also shown to participate in the epithelial-to-mesenchymal transition by regulating the alternative splicing of SF2/ASF. Sam68 was shown to regulate the activity-dependent alternative splicing of the neurexin-1 in the central nervous system with implications for neurodevelopment disorders. Sam68 influences alternative splicing of the mTOR kinase contributing to the lean phenotype observed in the Sam68 deficient mice.
The RNA binding activity of Sam68 is regulated by post-translational modifications such that Sam68 is often referred to as a STAR (Signal Transduction Activator of RNA) protein by which signals from growth factors or soluble tyrosine kinases, such as Src family kinases, act to regulate cellular RNA processes such as alternative splicing. For example, the Sam68-dependent CD44 alternative splicing of exon v5 is regulated by ERK phosphorylation of Sam68 and Bcl-x alternative splicing is regulated by the p59fyn-dependent phosphorylation of Sam68. Sam68 is also downstream of the epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF)/Met receptor (c-Met), leptin and tumor necrosis factor (TNF) receptors. While the role of Sam68 in these pathways is slowly emerging much remains to be determined. Sam68 has also been shown to re-localize in the cytoplasm near the plasma membrane, where it functions to transport and regulate the translation of certain mRNAs and regulates cell migration.
Gene knockout studies
Sam68-deficient mice were generated by targeted disruption of exons 4-5 of the sam68 gene, which encode the functional region of the KH domain. The genotypes of the offspring from heterozygote intercrosses exhibited a Mendelian segregation at E18.5. Despite the lack of visible deformity, many of the Sam68-/- pups died at birth of unknown causes. Sam68+/- mice were phenotypically normal and Sam68-/- pups that survived the peri-natal period invariably lived to old age. Sam68-/- mice weighed less than Sam68+/+ littermates and magnetic resonance imaging analysis confirmed that young Sam68-/- mice exhibited a profound reduction in adiposity, although food intake was similar. Moreover, Sam68-/- mice were protected against dietary-induced obesity. Sam68 deficient preadipocytes (3T3-L1 cells) had impaired adipogenesis and Sam68-/- mice had ~45% less adult derived stem cells (ADSCs) in their stromal vascular fraction (SVF) from WAT.
Sam68-/- mice did not develop tumors and showed no immunological or other major illnesses. Sam68-/- mice did, however, have difficulty breeding due to male infertility and female subfertility. The Sam68-null mice exhibited motor coordination defects and fell from the rotating drum at lower speeds and prematurely compared to the wild-type controls. Sam68-/- mice are protected against age-induced osteoporosis. Using the mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) mouse model of mammary tumorigenesis, it was shown that reduced Sam68 expression decreases tumor burden and metastasis. Kaplan-Meier curves showed that loss of one sam68 allele (PyMT; Sam68+/-) was associated with a significant delay in the onset of palpable tumors and a significant reduction in tumor multiplicity. These findings suggest that Sam68 is required for PyMT-induced mammary tumorigenesis. The knockdown of Sam68 expression in PyMT-derived mammary cells reduced the number of lung tumor foci in athymic mice, suggesting that Sam68 is also required for mammary tumor metastasis. The knockdown of Sam68 delayed LNCaP prostate cancer cells proliferation. The roles of Sam68 in cancer have been reviewed.
- Wong G, Muller O, Clark R, Conroy L, Moran MF, Polakis P, McCormick F (Jun 1992). "Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62". Cell. 69 (3): 551–8. doi:10.1016/0092-8674(92)90455-L. PMID 1374686.
- Lee J, Burr JG (Jan 2000). "Salpalpha and Salpbeta, growth-arresting homologs of Sam68". Gene. 240 (1): 133–47. doi:10.1016/S0378-1119(99)00421-7. PMID 10564820.
- "Entrez Gene: KH domain containing, RNA binding, signal transduction associated 1".
- Galarneau A, Richard S (May 2009). "The STAR RNA binding proteins GLD-1, QKI, SAM68 and SLM-2 bind bipartite RNA motifs". BMC Mol Biol. 10 (47): 47. doi:10.1186/1471-2199-10-47. PMC 2697983. PMID 19457263.
- Lin Q, Taylor SJ, Shalloway D (Oct 1997). "Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains". J Biol Chem. 272 (43): 27274–27280. doi:10.1074/jbc.272.43.27274. PMID 9341174.
- Cheng C, Sharp PA (Jan 2006). "Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion". Mol Cell Biol. 26 (1): 362–70. doi:10.1128/MCB.26.1.362-370.2006. PMC 1317625. PMID 16354706.
- Matter N, Herrlich P, Konig H (Dec 2002). "Signal-dependent regulation of splicing via phosphorylation of Sam68". Nature. 420 (6916): 691–5. doi:10.1038/nature01153. PMID 12478298.
- Paronetto MP, Achsel T, Massiello A, Chalfant CE, Sette C (Mar 2007). "The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x". J Cell Biol. 176 (7): 929–39. doi:10.1083/jcb.200701005. PMC 2064079. PMID 17371836.
- Pedrotti S, Bielli P, Paronetto MP, Ciccosanti F, Fimia GM, Stamm S, Manley JL, Sette C (Apr 2010). "The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy". EMBO J. 29 (7): 1235–47. doi:10.1038/emboj.2010.19. PMC 2857462. PMID 20186123.
- Chawla G, Lin CH, Han A, Shiue L, Ares MJ, Black DL (Jan 2009). "Sam68 regulates a set of alternatively spliced exons during neurogenesis". Mol Cell Biol. 29 (1): 201–13. doi:10.1128/MCB.01349-08. PMC 2612485. PMID 18936165.
- Iijima T, Wu K, Witte H, Hanno-Iijima Y, Glatter T, Richard S, Scheiffele P (Dec 2011). "SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1". Cell. 147 (7): 1601–14. doi:10.1016/j.cell.2011.11.028. PMC 3246220. PMID 22196734.
- Huot ME, Vogel G, Zabarauskas A, Ngo CT, Coulombe-Huntington J, Majewski J, Richard S (Apr 2012). "The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis". Mol Cell. 46 (2): 187–99. doi:10.1016/j.molcel.2012.02.007. PMID 22424772.
- Richard S (2010). "Reaching for the stars: Linking RNA binding proteins to diseases". Adv Exp Med Biol. 693: 142–57. PMID 21189691.
- Huot ME, Vogel G, Richard S (Nov 2009). "Identification of a Sam68 ribonucleoprotein complex regulated by epidermal growth factor". J Biol Chem. 284 (46): 31903–13. doi:10.1074/jbc.M109.018465. PMC 2797261. PMID 19762470.
- Locatelli A, Lange CA (Jun 2011). "Met receptors induce Sam68-dependent cell migration by activation of alternate extracellular signal-regulated kinase family members". J Biol Chem. 286 (24): 21062–72. doi:10.1074/jbc.M110.211409. PMC 3122167. PMID 21489997.
- Maroni P, Citterio L, Piccoletti R, Bendinelli P (Oct 2009). "Sam68 and ERKs regulate leptin-induced expression of OB-Rb mRNA in C2C12 myotubes". Mol Cell Endocrinol. 309 (1–2): 26–31. doi:10.1016/j.mce.2009.05.021. PMID 19524014.
- Ramakrishnan P, Baltimore D (Jul 2011). "Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor". Mol Cell. 43 (2): 167–79. doi:10.1016/j.molcel.2011.05.007. PMC 3142289. PMID 21620750.
- Paronetto MP, Messina V, Bianchi E, Barchi M, Vogel G, Moretti C, Palombi F, Stefanini M, Geremia R, Richard S, Sette C (Apr 2009). "Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis". J Cell Biol. 185 (2): 235–49. doi:10.1083/jcb.200811138. PMC 2700383. PMID 19380878.
- Richard S, Torabi N, Franco GV, Tremblay GA, Chen T, Vogel G, Morel M, Cleroux P, Forget-Richard A, Komarova S, Tremblay ML, Li W, Li A, Gao YJ, Henderson JE (Dec 2005). "Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss". PLoS Genet. 1 (6): e74. doi:10.1371/journal.pgen.0010074. PMC 1315279. PMID 16362077.
- Bianchi E, Barbagallo F, Valeri C, Geremia R, Salustri A, De Felici M, Sette C (Dec 2010). "Ablation of the Sam68 gene impairs female fertility and gonadotropin-dependent follicle development". Hum Mol Genet. 19 (24): 4886–94. doi:10.1093/hmg/ddq422. PMID 20881015.
- Lukong KE, Richard S (Jun 2008). "Motor coordination defects in mice deficient for the Sam68 RNA-binding protein". Behav Brain Res. 189 (2): 357–63. doi:10.1016/j.bbr.2008.01.010. PMID 18325609.
- Richard S, Vogel G, Huot ME, Guo T, Muller WJ, Lukong KE (Jan 2008). "Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis". Oncogene. 27 (4): 548–56. doi:10.1038/sj.onc.1210652. PMID 17621265.
- Busà R, Paronetto MP, Farini D, Pierantozzi E, Botti F, Angelini DF, Attisani F, Vespasiani G, Sette C (Jun 2007). "The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells". Oncogene. 26 (30): 4372–82. doi:10.1038/sj.onc.1210224. PMID 17237817.
- Bielli P, Busà R, Paronetto MP, Sette C (Jul 2011). "The RNA-binding protein Sam68 is a multifunctional player in human cancer". Endocr Relat Cancer. 18 (4): R91–R102. doi:10.1530/ERC-11-0041. PMID 21565971.
- Najib S, Martín-Romero C, González-Yanes C, Sánchez-Margalet V (2005). "Role of Sam68 as an adaptor protein in signal transduction". Cell. Mol. Life Sci. 62 (1): 36–43. doi:10.1007/s00018-004-4309-3. PMID 15619005.
- Koch CA, Moran MF, Anderson D, et al. (1992). "Multiple SH2-mediated interactions in v-src-transformed cells". Mol. Cell. Biol. 12 (3): 1366–74. doi:10.1128/mcb.12.3.1366. PMC 369570. PMID 1545818.
- Weng Z, Thomas SM, Rickles RJ, et al. (1994). "Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains". Mol. Cell. Biol. 14 (7): 4509–21. PMC 358823. PMID 7516469.
- Taylor SJ, Anafi M, Pawson T, Shalloway D (1995). "Functional interaction between c-Src and its mitotic target, Sam 68". J. Biol. Chem. 270 (17): 10120–4. doi:10.1074/jbc.270.17.10120. PMID 7537265.
- Richard S, Yu D, Blumer KJ, et al. (1995). "Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1". Mol. Cell. Biol. 15 (1): 186–97. PMC 231932. PMID 7799925.
- Nunès JA, Truneh A, Olive D, Cantrell DA (1996). "Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules". J. Biol. Chem. 271 (3): 1591–8. doi:10.1074/jbc.271.3.1591. PMID 8576157.
- Vadlamudi RK, Joung I, Strominger JL, Shin J (1996). "p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins". J. Biol. Chem. 271 (34): 20235–7. doi:10.1074/jbc.271.34.20235. PMID 8702753.
- Finan PM, Hall A, Kellie S (1996). "Sam68 from an immortalised B-cell line associates with a subset of SH3 domains". FEBS Lett. 389 (2): 141–4. doi:10.1016/0014-5793(96)00552-2. PMID 8766817.
- Bunnell SC, Henry PA, Kolluri R, et al. (1996). "Identification of Itk/Tsk Src homology 3 domain ligands". J. Biol. Chem. 271 (41): 25646–56. doi:10.1074/jbc.271.41.25646. PMID 8810341.
- Andreotti AH, Bunnell SC, Feng S, et al. (1997). "Regulatory intramolecular association in a tyrosine kinase of the Tec family". Nature. 385 (6611): 93–7. doi:10.1038/385093a0. PMID 8985255.
- Trüb T, Frantz JD, Miyazaki M, et al. (1997). "The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling". J. Biol. Chem. 272 (2): 894–902. doi:10.1074/jbc.272.2.894. PMID 8995379.
- Lawe DC, Hahn C, Wong AJ (1997). "The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68". Oncogene. 14 (2): 223–31. doi:10.1038/sj.onc.1200821. PMID 9010224.
- Barlat I, Maurier F, Duchesne M, et al. (1997). "A role for Sam68 in cell cycle progression antagonized by a spliced variant within the KH domain". J. Biol. Chem. 272 (6): 3129–32. doi:10.1074/jbc.272.6.3129. PMID 9013542.
- Fusaki N, Iwamatsu A, Iwashima M, Fujisawa J (1997). "Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling". J. Biol. Chem. 272 (10): 6214–9. doi:10.1074/jbc.272.10.6214. PMID 9045636.
- Guinamard R, Fougereau M, Seckinger P (1997). "The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS". Scand. J. Immunol. 45 (6): 587–95. doi:10.1046/j.1365-3083.1997.d01-447.x. PMID 9201297.
- Resnick RJ, Taylor SJ, Lin Q, Shalloway D (1997). "Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis". Oncogene. 15 (11): 1247–53. doi:10.1038/sj.onc.1201289. PMID 9315091.
- Chen T, Damaj BB, Herrera C, et al. (1997). "Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain". Mol. Cell. Biol. 17 (10): 5707–18. PMC 232419. PMID 9315629.
- Tang J, Feng GS, Li W (1997). "Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor". Oncogene. 15 (15): 1823–32. doi:10.1038/sj.onc.1201351. PMID 9362449.
- Sung CK, Choi WS, Sanchez-Margalet V (1998). "Guanosine triphosphatase-activating protein-associated protein, but not src-associated protein p68 in mitosis, is a part of insulin signaling complexes". Endocrinology. 139 (5): 2392–8. doi:10.1210/en.139.5.2392. PMID 9564850.