Through expression of EGFP-tagged RIG1 variants, we have identified that the NC domain, especially the NC motif, plays the most profound role in the RIG1-mediated growth inhibition and cellular apoptosis of HtTA cells. The conclusion is supported first by significantly decreased the effect of RIG1 on cell death of the fusion protein (RIG1Δ40–136) with deletion at the NC domain, and fusion proteins that contain only the NC domain (RIG1/NC) exhibit profound proapoptotic activity. In addition, we demonstrated that the RIG1111–123 within the NC domain increases cell death of HtTA cells, and that mutation of the NC motif results in complete abolishment of this activity. The importance of the NC motif is further demonstrated by the significant decrease of cell death in cells expressing the recombinant RIG1 variants that have two (NC) or three (NCR) amino acid mutations. Significance of the NC motif in RIG1 function is supported by the evolutionary conservation of the motif [6, 7]. Although a decrease of RIG1 expression was frequently observed in several types of cancer tissues [24–27], no mutation at the NC motif of RIG1 has been reported.
Analysis of the structure/function relationship of the NC motif and adjacent amino acids has been very limited. Mondal and coworkers first demonstrated that both cysteine residues within the DP of LRAT (LRAT159–171) (Fig 3B) are important for its enzymatic activity, and that the cysteine in the NC motif is the critical active-site involved in catalysis . Consistent with previous observations that demonstrate proapoptotic activities of DPs derived from HREV107 and RIG1 in melanoma cells (Fig 4B) , we show that RIG1111–123 induces cell death when introduced into HtTA cervical cancer cells as either a FITC-conjugated peptide or an EGFP fusion protein. In contrast to the significant role of Leu120 in RIG1111–123-mediated growth inhibition of E:WM-115 melanoma cells , this study demonstrated that the NC motif is indispensable, the Arg121 residue plays a minor role and the Leu120 has no effect on the anti-proliferative activities of RIG1111–123 in HtTA cells. This conclusion is supported first by FG for NC substitution which resulted in complete or significantly loss of anti-proliferative activities of FITC-RIG1111–123 or EGFP-RIG1111–123 respectively, second by profound loss of anti-proliferative activities in NC or NCR mutated RIG1 fusion proteins, third by reduced proapoptotic activities of the Arg121mutated RIG1111–123 and RIG1, and fourth by the lack of effect of Leu120 mutation on proapoptotic activities induced by RIG1111–123 or RIG1. In addition to HtTA cervical cancer cells, results from this and previous  studies also show growth suppressive activities of RIG1111–123 on established or primary culture of melanoma cells but not normal human fibroblasts and proliferating melanocytes. Therefore, RIG1111–123 is selectively toxic to tumor cells, and the significance of Leu120 in the RIG1111–123-mediated growth suppression shown previously in melanoma cells is tumor cell type specific.
RIG1111–123 is predicted to have an alpha helical structure [6, 22], and selectively binds to cis-binding elements and activates transcription factors involved in the G1/G0 → S transition in melanoma cells . In this study, we demonstrated that RIG1111–123 is targeted to nuclei of sensitive HtTA and A2058 cells, and mutations that render the loss of growth inhibitory activities result in cytoplasmic distribution of the DPs. Also, RIG1111–123 that has no effect on normal fibroblasts is distributed only within the cytoplasm. The tight association between nuclear localization and growth suppressive activities of RIG1111–123 supports the notion that RIG1111–123-mediated cell death in sensitive tumor cells is likely to be mediated through sequence-specific DNA binding that leads to alteration in gene expression involved in the cell cycle and apoptosis. Absence of nuclear localization of RIG1111–123 in resistant melanoma cells  and normal human fibroblasts may be related to failure in nuclear trafficking or to retention of the peptide. The α-helical structure of RIG1111–123 within the EGFP-RIG1111–123 fusion protein is mostly maintained predicted using SSpro and PSIPREDView programs. This supports the growth suppressive activity of EGFP-RIG1111–123. In contrast to complete loss of activities in NC- or NCR-mutated DPs, growth suppressive activities of the NC- or NCR-mutated EGFP-DP fusion proteins were significantly, but not completely, reduced. The increase in nuclear localization of the double and triple mutated EGFP-DPs, as compare to that of FITC-conjugated DPs, may be related to partial growth suppressive activities of the mutated EGFP-DPs. Currently, critical amino acids involving the binding of RIG1111–123 to cis transcription elements have not been addressed, although RIG1111–123 has been shown to bind cis binding elements for receptors of retinoic acids . The fact that nuclear distribution of RIG1111–123 was completely disrupted by mutations at the NC motif or Arg121, and was only partially altered by Leu120 mutation suggests that the NC motif and Arg121 play a profound role and Leu120 a minor role in the binding of RIG1111–123 to cis-elements. In addition to the four amino acids investigated in this study, six amino acids (EHFV–YG) within RIG1111–123 are conserved across the HREV107 protein family and LRAT (Fig 3A). Further analysis using site-directed DP mutants will be useful in dissecting the role of the NC motif, Arg121, Leu120 and other six conserved amino acids in the DNA binding properties as well as regulation of promoter and growth suppressive activities of RIG1111–123.
RIG1ΔN4 and RIG1ΔC are different only by 20 amino acids at the N-terminus, which consist of the proline-rich region. RIG1ΔN4 that is devoid of the proline-rich region inhibits growth and induces apoptosis of HtTA cells. However, RIG1ΔC fails to have effect on cell growth, apoptosis and activation of Ras and tissue transglutaminase I when the truncated protein is expressed as fusion proteins that have myc or EGFP tagged to the N- or C-terminus [10, 12, 13, 18, 19]. These results suggest that the N-terminal proline-rich region of RIG1 may bind effectors that prevent RIG1ΔC from exerting effects on the regulation of cell growth and apoptosis. Truncation of the N-terminal region, as demonstrated in RIG1ΔN4, restores these activities. In fact, the proline-rich region of HREV107 has recently been shown to bind a subunit of the protein phosphatase 2A, PR65α. The binding leads to inhibiting the enzyme's activity and is important for proapoptotic activities of HREV107 on ovarian cancer cells . Identification of a binding protein for the proline-rich region of RIG1 will be important to delineate the functional domain at the extreme N-terminus of RIG1.
Most results from previous studies demonstrate perinuclear and endomembrane localization of transfected wild type RIG1, HREV107 and HRASLS, which induce cellular apoptosis and/or differentiation of normal and cancer cells [9, 12–14, 18]. However, nuclear localization of HREV107 and RIG1 was also observed in fibroblasts and cancer cells, and cytoplasmic but not nuclear targeted HREV107 is related to growth stimulation and poor patient survival of non-small cell lung cancer [11, 14, 24]. Therefore, proteins of this gene family may localize to multiple intracellular compartments where they have distinct activities. Previous studies in HtTA cells and keratinocytes have demonstrated that RIG1-induced apoptosis or differentiation-related apoptosis is mediated through Golgi apparatus or mitochondria via caspase 3-dependent and/or -independent mechanisms [12, 13]. Similarly, caspase 3-mediated apoptosis is reported in keratinocytes expressing RIG1 with 124 amino acids truncated at the N-terminus . Results of this study in HtTA cells also demonstrated activation of caspase 3 during apoptosis induced by variants (RIG1ΔN1, RIG1ΔN2 and RIG1ΔN3) that contain the C-terminal hydrophobic domain but not RIG1ΔN4 and RIG1/NC that are deleted at both N- and C-termini. Although caspase 3 activation was observed in RIG1ΔN1, RIG1ΔN2 and RIG1ΔN3, RIG1ΔN1 and RIG1ΔN2 are mainly distributed in the nucleus whereas RIG1ΔN3 is in the perinuclear region. Therefore, presence of C-terminal hydrophobic domain rather than subcellular localization appears to play an important role in the caspase 3 activation during apoptosis induced by N-terminal truncated RIG1 variants. The nuclear targeted RIG1111–123 that exhibits DNA binding and proapoptotic activities was first demonstrated by Simmons et al. . This study further demonstrated that proapoptotic activities of truncated RIG1 variants, RIG1ΔN4 and RIG1/NC, and the nuclear targeted RIG1111–123 are mediated through the caspase 3-independent mechanism in cervical cancer cells. RIG1ΔN4 and RIG1/NC were found in both nucleus and cytoplasm. Whether the mechanism of apoptosis induced by RIG1ΔN4 and RIG1/NC is similar to that of RIG1111–123 that is mediated through regulation of gene expression, as proposed by Simmons et al , needs further investigation.
Wild type RIG1 and N-terminal truncated variants such as RIG1ΔN1, RIG1ΔN2 and RIG1ΔN3 were expressed at much lower levels than RIG1ΔN4 and RIG1/NC that were truncated at both N- and C-termini. Short protein half life  or an increase in distribution to the particulate fraction due to N-terminal truncation  may be responsible for the low protein levels observed in N-terminal truncated RIG1 variants. Although expressed at low levels, the extent of growth suppression on HtTA cells induced by N-terminal truncated variants was relatively similar to the RIG1ΔN4 and RIG1/NC. Similar potent proapoptotic activities, despite being expressed at extremely low levels, of RIG1 variants with N-terminal truncation for 112 to 124 amino acids were also reported in keratinocytes .
RIG1 is a growth regulator that suppresses cell growth through induction of cellular differentiation and/or apoptosis. The differentiation-inducing activity of RIG1 on keratinocytes is mediated through the activation of tissue transglutaminase I [13, 17]. A structure/function analysis has demonstrated that the N-terminal region of RIG1 is required for pro-differentiation activities, regardless, the C-terminal hydrophobic region by itself is enough for the activation of tissue transglutaminase I . Recently, results from our [12, 19] and others  studies in HtTA cervical and ovarian cancer cells have demonstrated that RIG1 inhibits expression and activation of signaling molecules such as HER2, RAS, PI3K/AKT and mTOR that are involved in the regulation of cell growth, apoptosis and tumor invasion. Further analysis using various truncated and mutated RIG1 variants will be useful to dissect mechanisms as well as functional domains and critical motifs of RIG1 in the regulation of signal pathways involving growth factor receptors.