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 ERKphosphorylation 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.
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.
^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 Chem272 (43): 27274–27280. doi:10.1074/jbc.272.43.27274. PMID9341174.
^ abcdHuot 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 Cell46 (2): 187–99. doi:10.1016/j.molcel.2012.02.007. PMID22424772.
^Richard S (2010). "Reaching for the stars: Linking RNA binding proteins to diseases". Adv Exp Med Biol693: 142–57. PMID21189691.
^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 Genet19 (24): 4886–94. doi:10.1093/hmg/ddq422. PMID20881015.
^Richard S, Vogel G, Huot ME, Guo T, Muller WJ, Lukong KE (Jan 2008). "Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis". Oncogene27 (4): 548–56. doi:10.1038/sj.onc.1210652. PMID17621265.
^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". Oncogene26 (30): 4372–82. doi:10.1038/sj.onc.1210224. PMID17237817.
^Bielli P, Busà R, Paronetto MP, Sette C (Jul 2011). "The RNA-binding protein Sam68 is a multifunctional player in human cancer". Endocr Relat Cancer18 (4): R91–R102. doi:10.1530/ERC-11-0041. PMID21565971.
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. PMID15619005.
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. PMID8576157.
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. PMID8702753.
Andreotti AH, Bunnell SC, Feng S, et al. (1997). "Regulatory intramolecular association in a tyrosine kinase of the Tec family.". Nature385 (6611): 93–7. doi:10.1038/385093a0. PMID8985255.
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. PMID8995379.
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.". Oncogene14 (2): 223–31. doi:10.1038/sj.onc.1200821. PMID9010224.
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. PMID9013542.
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. PMID9045636.
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.". Oncogene15 (15): 1823–32. doi:10.1038/sj.onc.1201351. PMID9362449.
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.". Endocrinology139 (5): 2392–8. doi:10.1210/en.139.5.2392. PMID9564850.