hnRNPA 1 couples nuclear export and translation of specific mRNAs downstream of FGF-2 / S 6 K 2 signalling

The increased cap-independent translation of antiapoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylationdependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its reimport into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCLXL expression. In short, phosphorylation on novel Nterm sites of hnRNPA1 promotes translation of antiapoptotic proteins and is indispensable for the prosurvival effects of FGF-2. INTRODUCTION Deregulation of apoptotic cell death is a hallmark of cancer and is involved in the development of resistance to therapy. This is a leading cause of fatalities from common cancers including lung cancer. Several proteins can mediate cell death resistance (1–3) including fibroblast growth factor 2 (FGF2) (4–6). Indeed, we have shown that FGF-2 signalling leads to the assembly of a multi-protein complex comprising BRaf, protein kinase C and ribosomal S6 kinase 2 (S6K2) but not S6K1. The activation of S6K2 occurs in a stepwise manner initiated by phosphorylation of the three proline-directed serines in the auto-inhibitory domain, Ser410, Ser-417 and Ser-423 downstream of MEK/ERK signalling. Subsequently, phosphorylation of Ser-370 then enables phosphorylation of Thr-388 by the mTORC1 complex followed by that of Thr-228 by PDK1 (7). Activated S6K2 then enhances the translation of anti-apoptotic proteins such as BCL-XL and X chromosome-linked inhibitor of apoptosis (XIAP) (4). Translation of these mRNAs under conditions of cellular stress including anti-cancer therapies is mediated by an internal ribosomal entry site (IRES) located in the 5′ untranslated region (UTR) (8–10). However, we only have limited understanding of the modulators of IRES-based translation downstream of S6K2 activation (11). S6K2 shuttles between the nucleus and the cytoplasm in response to growth factor signalling. In addition to its diffuse nuclear localization, a proportion of S6K2, but not S6K1, has been shown to co-localize with CTR453 and tubulin at the level of the centrosome (7). *To whom correspondence should be addressed. Tel: +44 20 3311 1421; Fax: +44 203 313 5830; Email: m.seckl@imperial.ac. uk Correspondence may also be addressed to Dr Olivier Pardo. Tel: +44 207 594 2814; Fax: +44 203 313 5830; Email: o.pardo@imperial.ac. uk Disclaimer: The funders have no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. C © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D ow naded rom http/academ ic.p.com /nar/article-a42/20/12483/2903192 by gest on 24 Jauary 2019 12484 Nucleic Acids Research, 2014, Vol. 42, No. 20 The hnRNP family comprises at least 20 members termed hnRNPA1 to hnRNPU (12). These proteins are mainly located in the nucleus, associated with pre-mRNA to influence their splicing, metabolism and transport with some hnRNPs reported to shuttle between the nucleus and the cytoplasm (12). Among these, hnRNPA1 was shown to contain a novel nuclear localisation signal, called M9, which enables both its nuclear entry and exit (13). Interestingly, interfering with hnRNPA1 shuttling, and the resulting accumulation of this protein in the cytoplasm, prevents BCL-XL and XIAP expression in several cell systems (9,10,14). Moreover, when hnRNPA1 is bound to these mRNAs, it suppresses IRESmediated translation (9,10,15–17). However, the regulation of this hnRNPA1 activity downstream of cellular signalling is not understood. Here, we identified hnRNPA1 as a distinct substrate for S6K2 downstream of FGF-2 signalling that is crucial for the anti-apoptotic function of this pathway. We found that FGF-2 signalling promotes the cycling of hnRNPA1 between the nucleus and the cytoplasm through a series of stepwise post-translational modifications. FGF-2 stimulation and S6K2 activation lead first to the phosphorylation of hnRNPA1 on a novel site (Ser4) that promotes the binding of this protein to BCL-XL and XIAP mRNAs. Then, following association with the export factor Nxf-1, hnRNPA1/mRNA complexes are exported from the nucleus in a MEK-dependent fashion. Finally, once in the cytoplasm, phosphorylated hnRNPA1 interacts with 14-3-3 and leading to its sumoylation on K183. This last event is necessary for the re-import of hnRNPA1 and subsequent shuttling cycles as sumo-deficient mutants for this site accumulate in the cytoplasm. In short, our work demonstrates a previously undescribed mechanism through which FGF2 signalling increases the export of BCL-XL and XIAP mRNAs out of the nucleus and promotes their subsequent translation. Targeting the enzymes that regulate these posttranslational modifications may provide a novel and efficient way to silence this pro-survival signalling mechanism in the treatment of cancer. MATERIALS AND METHODS Reagents and antibodies See Supplementary Materials.