Sequestration of RBM10 in Nuclear Bodies: Targeting Sequences and Biological Significance.

RBM10 is an RNA-binding protein that regulates alternative splicing (AS). It localizes to the extra-nucleolar nucleoplasm and S1-1 nuclear bodies (NBs) in the nucleus. We investigated the biological significance of this localization in relation to its molecular function. Our analyses, employing deletion mutants, revealed that RBM10 possesses two S1-1 NB-targeting sequences (NBTSs), one in the KEKE motif region and another in the C2H2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C2H2 ZnF not only acts as an NBTS, but is also essential for AS regulation by RBM10. Moreover, RBM10 does not participate in S1-1 NB formation, and without alterations of RBM10 protein levels, its NB-localization changes, increasing as cellular transcriptional activity declines, and vice versa. These results indicate that RBM10 is a transient component of S1-1 NBs and is sequestered in NBs via its NBTSs when cellular transcription decreases. We propose that the C2H2 ZnF exerts its NB-targeting activity when RBM10 is unbound by pre-mRNAs, and that NB-localization of RBM10 is a mechanism to control its AS activity in the nucleus.

Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705-SH2 through C-terminal tail modulation.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.

RBM10 regulates centriole duplication in HepG2 cells by ectopically assembling PLK4-STIL complexes in the nucleus.

RNA-binding motif protein 10 (RBM10) primarily regulates alternative splicing of certain genes. Loss-of-function mutations in RBM10 have been frequently reported in patients with various cancers. However, how RBM10 levels affect cell proliferation and tumorigenesis remains unknown. To elucidate the role of RBM10 in cell proliferation, we established HepG2-RBM10 knockout cell lines and derivative doxycycline-inducible RBM10-expressing cells. RBM10 over-expression caused growth arrest in the M phase with a monopolar spindle because of impaired centriole duplication. Two RBM10 splicing mutants, one with F345A/F347A and the other with only the C-terminal half (401-930), were sufficient to cause growth arrest, whereas an RBM10 mutant with cytoplasmic localization forced by an NES did not show growth arrest. RBM10 over-expression induced the formation of many large nuclear domains containing RBM10, PLK4, STIL and SAS6, which are the regulatory proteins involved in centriole duplication. Consistently, the centrioles in the RBM10-over-expressing HepG2 cells lost PLK4 and STIL, accounting for the unsuccessful centriole duplication. In contrast, RBM10 depletion resulted in elevated levels of cytoplasmic PLK4 with a concomitant increase in the number of centrioles in HepG2 cells but not in A549 cells. Thus, nuclear RBM10 regulates normal chromosomal division in a cell-type-specific manner, independent of alternative RNA splicing.

The Molecular Basis of Chemical Chaperone Therapy for Oculocutaneous Albinism Type 1A.

Oculocutaneous albinism (OCA) is an autosomal recessive disease characterized by the reduction or complete lack of melanin pigment in the skin, hair, and eyes. No effective treatment for OCA is available at present. OCA type 1 is caused by mutations that disrupt the function of tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis. Recently, it was shown that tyrosinase in some patients with OCA type 1 mutation is retained in the endoplasmic reticulum and that its catalytic activity is lost, a phenomenon known as endoplasmic reticulum retention. However, to our knowledge, the intracellular localization of tyrosinase in Japanese patients with OCA type 1 missense mutations has not been reported. In this study, we first investigated the intracellular localization of Japanese OCA type 1A missense mutant tyrosinases using Western blotting and immunohistochemical staining. R77Q, R239W, D383N, and P431L mutant tyrosinases were retained in the endoplasmic reticulum, and H211Y mutant tyrosinase was partially transported to the Golgi apparatus. Second, we explored the possibility of chemical chaperone therapy for Japanese patients with OCA type 1A missense mutations and found that HeLa cells expressing P431L mutant tyrosinase have restored tyrosinase activity after treatment with a low-dose tyrosinase inhibitor, as a chemical chaperone, in a dose-dependent manner. These results provide the basis for a possible chemical chaperone therapy to recover tyrosinase activities in patients with OCA type 1A patients.

S1-1/RBM10: multiplicity and cooperativity of nuclear localisation domains.

BACKGROUND INFORMATION: S1-1, also called RBM10, is an RNA-binding protein of 852 residues. An alteration of its activity causes TARP syndrome, a severe X-linked disorder with pre- or post-natal lethality in affected males. Its molecular function, although still largely unknown, has been suggested to be transcription and alternative splicing. In fact, S1-1 localises in the nucleus in tissue cells and cultured cells. RESULTS: By deletion and substitution mutagenesis, a classical 17-amino-acid (aa) nuclear localisation sequence (NLS1) was identified at aa 743-759 in the C-terminal region of S1-1. NLS1 was bipartite, with its N-terminal basic cluster weakly contributing to the NLS activity. S1-1 contained two additional NLSs. One was in the aa 60-136 RNA recognition motif region (NLS2), and the other was a novel NLS motif sequence in the aa 481-540 octamer-repeat (OCRE) region (NLS3). The OCRE is a domain known to be critical in splicing regulation, as shown with RBM5, a close homologue of RBM10 [Bonnal et al. (2008) Mol. Cell 32, 81-95]. The NLS activities were verified by expressing each DNA sequence linked to EGFP or a FLAG tag. These multiple NLSs acted cooperatively, and S1-1 became completely cytoplasmic after the concomitant removal of all NLS domains. In some cell types, however, S1-1 was partly cytoplasmic, suggesting that cellular localisation of S1-1 is subjected to regulation. CONCLUSIONS: The present results indicate that S1-1 contains multiple NLSs that act cooperatively. Among them, the OCRE is a hitherto unreported NLS. The nuclear localisation of S1-1 appears to be regulated under certain circumstances. We discuss these NLSs in relation to the biochemical processes they are involved in.

IL-6-STAT3 signaling and premature senescence.

Cytokines play several roles in developing and/or reinforcing premature cellular senescence of young cells. One such cytokine, interleukin-6 (IL-6), regulates senescence in some systems in addition to its known functions of immune regulation and promotion of tumorigenesis. In this review, we describe recent advances in studies on the roles of IL-6 and its downstream signal transducer and activator of transcription 3 (STAT3) in regulating premature cellular senescence. IL-6/sIL-6Rα stimulation forms a senescence-inducing circuit involving the STAT3-insulin-like growth factor-binding protein 5 (IGFBP5) as a key axis triggering and reinforcing component in human fibroblasts. We describe how cytokines regulate the process of senescence by activating STAT3 in one system and anti-senescence or tumorigenesis in other systems. The roles of other STAT members in premature senescence also will be discussed to show the multiple mechanisms leading to cytokine-induced senescence.

The STAT3-IGFBP5 axis is critical for IL-6/gp130-induced premature senescence in human fibroblasts.

Cells undergo senescence in response to various conditions, including telomere erosion, oncogene activation and multiple cytokines. One of these cytokines, interleukin-6 (IL­6), not only functions in the immune system, but also promotes cellular senescence and cancer. Here we demonstrate that IL­6 and the soluble IL­6 receptor (sIL­6R) induce premature senescence in normal human fibroblasts by establishing a senescence-inducing circuit involving the signal transducer and activator of transcription 3 (STAT3) and insulin-like growth factor-binding protein 5 (IGFBP5). Stimulating TIG3 fibroblast cells with IL­6/sIL­6R sequentially caused an increase in reactive oxygen species (ROS) as early as day 1, followed by the DNA damage response, p53 accumulation and, finally, senescence on days 8-10. We found that STAT3 was required for the events leading to senescence, including the initial early-phase ROS increase and the induction of IL­1α/ß, IL­6 and CXCL8 mRNAs 4-5 d after IL­6/sIL­6R stimulation, suggesting that STAT3's role is indirect. We searched for STAT3-downstream molecule(s) responsible for the senescence-inducing activity in the supernatants of stimulated TIG3 and identified IGFBP5 as a major STAT3 mediator, because IGFBP5 was expressed from the early phase through the entire senescence process and was responsible for IL­6/STAT3-induced ROS increase and premature senescence. Thus, IL­6/sIL­6R forms a senescence-inducing circuit involving the STAT3-IGFBP5 axis as a key triggering and reinforcing component.

Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45.

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor. It is activated by cytokines, including interleukin-6 (IL-6) through phosphorylation at Tyr705 (pY705), which is required for its dimerization and nuclear translocation. However, the role of Ser727 phosphorylation, occurring during activation, remains poorly understood. Using a combination of HepG2-stat3-knockdown cells reconstituted with various STAT3 mutants and protein kinase inhibitors, we showed that phospho-S727 has an intrinsic mechanism for shortening the duration of STAT3 activity, in turn shortening the duration of socs3 mRNA expression. Both STAT3WT and STAT3Ser727Asp (S727D) but not STAT3Ser727Ala (S727A) showed rapid dephosphorylation of pY705 after the inhibition of tyrosine kinases. We found that the nuclear TC45 phosphatase is most likely responsible for the phospho-S727-dependent pY705 dephosphorylation because TC45 knockdown caused prolonged pY705 with sustained socs3 mRNA expression in STAT3WT but not in STAT3S727A, and overexpressed TC45 caused rapid dephosphorylation of pY705 in STAT3WT but not in STAT3S727A. We further showed that phospho-S727 did not affect the interaction of TC45 with STAT3, and that a reported methylation at K140 of STAT3 occurring after phospho-S727 was not involved in the pY705 regulation. These findings indicate that phospho-Ser727 determines the duration of STAT3 activity largely through TC45.

Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia.

BACKGROUND: Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth. OBJECTIVE: In this study, we examined the role and localization of laminin-332 and -511 in CIA. METHODS: We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment. RESULTS: Our data indicate that both laminin-332 and its receptor alpha 6 beta 4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of alpha 3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the alpha 3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA. CONCLUSIONS: We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels.

Spatiotemporal regulation of c-Fos by ERK5 and the E3 ubiquitin ligase UBR1, and its biological role.

c-Fos is regulated by phosphorylation and multiple turnover mechanisms. We found that c-Fos was ubiquitylated in the cytoplasm during IL-6/gp130 stimulation under MEK inhibition and sought the mechanisms involved in the regulation. We show that sustained ERK5 activity and the E3 ligase UBR1 regulate the stability and subcellular localization of c-Fos. UBR1, rapidly induced by STAT3, interacts with and ubiquitylates c-Fos in the cytoplasm for its accelerated degradation. ERK5 inhibits the nuclear export of c-Fos by phosphorylating Thr232 in the c-Fos NES(221-233) and disrupts the interaction of c-Fos with UBR1 by phosphorylating Ser32. Moreover, UBR1 depletion in HeLa cells, which constitutively express UBR1 at a high level, enhances both c-Fos expression and cell growth, whereas ERK5 depletion reduces both of them. Interestingly, an NES mutant of c-Fos, but not wild-type, substitutes ERK5 activity for HeLa cell proliferation. Thus, this spatiotemporal regulation of c-Fos by ERK5 and UBR1 is critical for the regulation of c-Fos/AP-1.

STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGFbeta-activated kinase 1 for STAT3 Ser-727 phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is activated by the IL-6 family of cytokines and growth factors. STAT3 requires phosphorylation on Ser-727, in addition to tyrosine phosphorylation on Tyr-705, to be transcriptionally active. In IL-6 signaling, the two major pathways that derive from the YXXQ and the YSTV motifs of gp130 cause Ser-727 phosphorylation. Here, we show that TGF-beta-activated kinase 1 (TAK1) interacts with STAT3, that the TAK1-Nemo-like kinase (NLK) pathway is efficiently activated by IL-6 through the YXXQ motif, and that this is the YXXQ-mediated H7-sensitive pathway that leads to STAT3 Ser-727 phosphorylation. Because NLK was recently shown to interact with STAT3, we explored the role of STAT3 in activating this pathway. Depletion of STAT3 diminished the IL-6-induced NLK activation by >80% without inhibiting IL-6-induced TAK1 activation or its nuclear entry. We found that STAT3 functioned as a scaffold for TAK1 and NLK in vivo through a region in its carboxyl terminus. Furthermore, the expression of the STAT3(534-770) region in the nuclei of STAT3-knockdown cells enhanced the IL-6-induced NLK activation in a dose-dependent manner but not the TGFbeta-induced NLK activation. TGFbeta did not cause STAT3 Ser-727 phosphorylation, even when the carboxyl region of STAT3 was expressed in the nuclei. Together, these results indicate that STAT3 enhances the efficiency of its own Ser-727 phosphorylation by acting as a scaffold for the TAK1-NLK kinases, specifically in the YXXQ motif-derived pathway.

Region 752-761 of STAT3 is critical for SRC-1 recruitment and Ser727 phosphorylation.

STAT3 regulates many target genes in response to cytokines and growth factors. To study the mechanisms of STAT3-dependent transcription, we established several cell lines in which HepG2-STAT3-knockdown cells were reconstituted with a variety of STAT3 mutants. Using these cell lines, we found that truncated STAT3(1-750), but not STAT3(1-761), could not recruit SRC-1/NcoA-1 and was not phosphorylated on Ser727. Furthermore, mutation of STAT3 L755 and F757 to alanines caused the loss of STAT3-dependent SRC-1 recruitment, leaving Ser727 phosphorylation intact. Consistent with this, the STAT3-L755A/F757A mutant showed no increase in acetylated histone H3 at Lys14 and a decreased level of RNA polymerase II recruited to the target gene promoter, although p300 recruitment and histone H4 acetylation were intact. This mutant also lost responsiveness to co-expressed SRC-1. Thus, the conserved STAT3 region from 752 to 761, called STAT3 CR2, plays critical roles in STAT3-dependent transcription by recruiting SRC-1 and allowing Ser727 phosphorylation.

Cytoplasmic c-Fos induced by the YXXQ-derived STAT3 signal requires the co-operative MEK/ERK signal for its nuclear translocation.

A STAT3 (signal transducer and activator of transcription 3)- and a MEK/Erk-mediated signal can be activated by cytokines, including IL-6 (interleukin-6), PDGF, and EGF. Recently, STAT3 and an ERK-signal were shown to co-operatively activate the c-fos gene. Activation of a truncated form of the IL-6 receptor subunit, gp130, that had only one YXXQ motif, induced both c-Fos and JunB in NIH3T3 cells through STAT3 without an apparent increase in the AP-1 (activator protein-1) activity. In contrast, concomitant stimulation of the STAT3 signal and a MEK/Erk-signal markedly increased AP-1 activity with enhanced c-Fos expression. Surprisingly, the c-Fos induced by the YXXQ-signal alone was localized to the cytoplasm, from which it translocated into the nucleus following TPA (12-O-tetradecanoyl-phorbol 13-acetate) treatment in a MEK/Erk-dependent manner. c-Fos that was expressed from a constitutive promoter localized to the nucleus and did not move into the cytoplasm in response to the YXXQ-signal. Rather, the YXXQ-signal was required during c-Fos production for it to be retained in the cytoplasm. Thus, the YXXQ-signal induces c-Fos expression through STAT3 and anchors the new c-Fos in the cytoplasm. In addition, the YXXQ-signal and an Erk signal co-operatively cause c-Fos activation in the nucleus.