Esse trabalho forneceu evidências para novos alvos ou reguladores da família de proteínas S6Ks, sendo estes principalmente: eIF2α e PARP1. As principais conclusões são:
Parte 1 (anexo I)
possíveis novos alvos das S6Ks foram identificados, permitindo estudos futuros que esclareçam novas funções dessas proteínas na via da mTOR;
2. A maior parte das proteínas de interação da S6K1 é citosólica, enquanto da S6K2 é nuclear;
3. S6K1 apresenta maior relevância em processos biológicos como resposta ao estresse, citoesqueleto, metabolismo, remodelamento de cromatina e fosfatases;
4. S6K2 apresenta maior relevância em processos biológicos como transcrição, biogênese de ribossomos, síntese protéica, replicação e reparo de DNA e splicing e processamento de RNA;
Parte 2:
5. Análises in silico mostraram que eIF2α apresenta o sítio predito de fosforilação de S6Ks, RXRXXT/S, o qual está extremamente conservado em diferentes espécies. Outras análises evidenciaram que as S6Ks apresentam score elevado para fosforilar o resíduo S58 da eIF2α;
6. Ensaios de imunoprecipitação revelaram que eIF2α é uma proteína de interação de S6K1 e S6K2;
7. A eIF2α apresenta resíduo anti-RXXT/S fosforilado na presença de insulina; 8. Ativação das S6Ks com insulina e FGF2 reduzem a fosforilação de eIF2α (S52). A ativação de S6Ks através de FGF2 se deu de maneira independente de mTORC1;
9. As formas constitutivamente ativas (CA) de S6K1 e S6K2 foram capazes de reduzir a fosforilação de eIF2α (S52), sendo que o CA de S6K2 também reduziu a expressão de ATG7, um marcador de autofagia;
10. Inibições farmacológicas de mTOR/S6Ks com rapamicina ou PF4708671 foram capazes de retomar a fosforilação de eIF2α (S52);
11. Mimetização de uma fosforilação no resíduo S58 através de uma mutação sítio dirigida (eIF2α S58E) modulou negativamente a fosforilação do resíduo S52, indicando uma regulação negativa entre esses dois resíduos de serina de eIF2α;
12. PARP1 parece co-imunoprecipitar com S6K2, mas não com S6K1;
As conclusões obtidas nesse projeto devem contribuir para um melhor entendimento da via mTOR/S6Ks e sua relação com a homeostase da célula. Não foi descartada a possibilidade de interação das S6Ks com as outras proteínas propostas inicialmente no trabalho, como a FXR1, PPM1B, PRMT5 e WDR77. Estudos futuros necessitam ser realizados para que haja um melhor entendimento dessas interações e de seus efeitos para a célula.
Como perspectivas deste estudo, experimentos adicionais ainda poderão ser realizados para melhor entendimento das interações das S6Ks com as proteínas eIF2α e PARP1. Para tanto, pretendemos ainda mostrar a interação dessas proteínas em situações com a presença de fatores de crescimento, estresse celular e inibição farmacológica da via mTOR/S6Ks. Uma vez que encontramos indícios que possa existir uma regulação negativa do estado de fosforilação de eIF2α no resíduo 52 quando fosforilada no resíduo 58 pelas S6Ks, pretende-se explorar mais esse aspecto realizando a técnica de CRISPR-Cas9, a qual será empregada com o objetivo de modificar o gene endógeno de eIF2α, mimetizando a fosforilação do resíduo 58 e analisando possíveis efeitos na regulação da autofagia e síntese protéica. Por fim, sabe-se que a fosforilação de eIF2α no resíduo 52 é vista aumentada em situações de obesidade e câncer, uma vez que o processo de estresse de retículo endoplasmático está desregulado. Dessa forma, utilizar estratégias que manipulem a fosforilação de eIF2α em seu resíduo serina 52 é interessante para uma melhor compreensão dos processos celulares que estão envolvidos com a obesidade e câncer.
O projeto de mestrado (processo nº 2015/00311-1) foi desenvolvido com o auxílio financeiro da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). As opiniões, hipóteses e conclusões ou recomendações expressas neste material são de responsabilidade do(s) autor(es) e não necessariamente refletem a visão da FAPESP.
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§C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2016, 00, 1–17 DOI 10.1002/pmic.201500249 1
RESEARCH ARTICLE
Different interactomes for p70-S6K1 and p54-S6K2
revealed by proteomic analysis
Isadora C. B. Pavan1, Sami Yokoo2, Daniela C. Granato2, Let´ıcia Meneguello3,
Carolina M. Carnielli2, Mariana R. Tavares1, Camila L. do Amaral1, Lidia B. de Freitas1,
Adriana F. Paes Leme2, Augusto D. Luchessi3 and Fernando M. Simabuco1
1 Laboratory of Metabolic Disorders, School of Applied Sciences, University of Campinas, Limeira, Sa˜ o Paulo,
Brazil
2 Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Sa˜ o
Paulo, Brazil
3 Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, Sa˜ o Paulo, Brazil
S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrom- etry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affin- ity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 inter- acting proteins were predominantly related to “cytoskeleton” and “stress response,” whereas p54-S6K2 interactome was more associated to “transcription,” “splicing,” and “ribosome bio- genesis.” Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2a, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.
Keywords:
Cell Biology / Immunoprecipitation / mTOR / S6K
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Received: June 25, 2015 Revised: June 28, 2016 Accepted: August 3, 2016
1 Introduction
Correspondence: Prof. Fernando Moreira Simabuco, Laborato´ rio de Distu´ rbios do Metabolismo, Faculdade de Cieˆ ncias Aplicadas, Universidade de Campinas, R. Pedro Zaccaria, 1300, sala LA 421, Jardim Sa˜ o Paulo, 13484-350 Limeira, Sa˜ o Paulo, Brazil
E-mail: simabuco@gmail.com
Abbreviations: CRAPome, contaminant repository for affinity purification; IIS, Integrated Interactome System; IMP1, insulin- like growth factor 2 mRNA-binding protein 1; mTORC1, mam- malian target of rapamycin complex 1; NCL, nucleolin; NPM1,
The S6K (ribosomal protein S6 kinase) proteins, includ- ing S6K1 and S6K2, are known effectors of the mTORC1 (mammalian target of rapamycin complex 1) pathway, which plays an important role in the cellular control of energy
nucleophosmin; SAINT, significance analysis of interactome; SG, stress granule; SP, SAINT probability
Colour Online: See the article online to view Figs. 1–6, and 8 in
colour.
2 I. C. B. Pavan et al. Proteomics 2016, 00, 1–17
homeostasis [1–3]. mTORC1 integrates signals derived from many environmental stimuli to promote anabolic functions and inhibit cell catabolism [1, 2].
Two genes of S6Ks are known, named RPS6KB1 and RPS6KB2, encoding two different proteins, called p70-S6K1 and p54-S6K2, respectively [4]. Each of these proteins have larger isoforms produced by alternative use of the AUG trans- lation initiation codon, called p85-S6K1 and p56-S6K2, which have an addition of a predicted nuclear localization signal at the N-terminus, suggesting that the localization of these iso- forms are predominantly nuclear [1, 3–5]. A study, however, has shown that the location of p85-S6K1 is cytoplasmic, while p70-S6K1 can be found in cytoplasm and nucleus [6]. The two S6K2 isoforms exhibit a nuclear localization signal at the C- terminus, which also suggests that they are localized in the nucleus [3, 7]. Furthermore, S6K2 proteins present a proline- rich domain at the C-terminus, allowing these isoforms to interact with SH3 (Src homology 3) and WW proteins do- mains [1]. Over the years, studies have exercised major efforts to discover new functions of the mTORC1/S6K1 signaling, revealing that this axis can control many cellular processes and is related to several diseases [1–3]. However, there is a limitation of studies on S6K2, turning it into a neglected S6K isoform [3, 5].
S6K1 is involved in metabolism regulation through the in- teraction with CAD [8], AMPK [9], IRS1 [10, 11], GSK3 [12], and PFK2 [13]. Other studies have shown that S6K1 regulates protein synthesis through interactions with S6 [14], eEF2K [15], PDCD4 [16], EIF4B [17], and FMRP [18]. In addition, there is an involvement of S6K1 in mRNA splicing process by interaction with CBP80 [19] and SKAR [20], as well as in in- flammatory process by interaction with TAK1 [21]. Processes such as cytoskeleton organization may also have involvement of S6K1 by interaction with CDC42 and RAC1 proteins [22]. Additionally, S6K1 also relates to the apoptotic process, due to interactions with BAD [23] and Mdm2 [24]. Besides, S6K1 is able to phosphorylate ERa [25] and cAMP-responsive ele- ment modulator (CREM) [26], and consequently to regulate transcription. Using phospho-proteomics approaches, stud- ies also identified CCTþ, GRP75, and PGK1, which may act as a chaperones, as new targets of S6K1 [27, 28].
Although there are few studies characterizing the inter- actome of S6K2 [3, 5], a recent study has shown that S6K2 presents some nuclear interaction partners of the hnRNPs family [29]. Another study has shown that S6K2, instead of S6K1, presents a localization close to centriole, suggesting a possible role in cell division [30]. It has been also demon- strated that S6K2 is able to phosphorylate histone H3, which may be related to transcriptional regulation [31]. Finally, stud- ies have shown that S6K2 knockout in mice leads to larger animals in comparison to smaller animals generated by S6K1 knockout [1, 10, 32].
The present study explored the different interactomes of p70-S6K1 and p54-S6K2, identifying new interacting proteins involved in the regulation of several cellular processes, such as “transcription,” “protein synthesis,” “splicing and RNA processing,” “stress response,” “inflammation and immu- nity,” “cytoskeleton organization,” “biogenesis of ribosomes,” “DNA replication and repair,” “chromatin remodeling,” and others. These proteins may represent new possible regula- tors or targets of S6Ks, promoting, in this way, advances in understanding the role of these kinases. Based on the com- parative interactome analysis of p70-S6K1 and p54-S6K2, we revealed putative different interaction partners, which may reflect differences in function and subcellular localization of these isoforms.
2 Material and methods
Cells and plasmids
HEK293 and HeLa cells were cultivated in DMEM supple- mented with 10% v/v FBS (fetal bovine serum). Cells were transfected using Lipofectamine PLUS Reagent or Lipofec- tamine 2000 (Thermo Scientific), following the manufac- turer’s instructions. For FLAG-tagged protein expression, a modified pcDNA 3.1 (+) (Thermo Scientific) was gener- ated cloning the FLAG peptide coding sequence upstream of the multiple cloning site, generating the pcDNA-FLAG vec- tor. The full-length S6K1 and S6K2 cDNA (accession num-