Characterization of the type 2A phosphatase regulatory protein, TIPRL and alpha4

Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As células respondem constantemente a uma enorme variedade de estímulos, que são interpretados e integrados por meio de redes de sinalização, dando origem a uma resposta biológica. Defeitos nesses circuitos são a causa de diversas doenças, incluindo muitos, se não todos os tipos de câncer. As fosfatases, enzimas que removem grupamentos fosfato dos substratos de quinases, dependem principalmente de subunidades regulatórias para definir sua especificidade. As fosfatases do tipo 2A constituem a subfamília PPP, que é formada por PP2A, PP4 e PP6. PP2A é a principal fosfatase solúvel de fosfosserina e fosfotreonina em células animais e é encontrada predominantemente como uma holoenzima formada por uma subunidade catalítica (C), uma subunidade regulatória (B, B', B'' ou B''') e uma de ancoragem (PR65/A). Em levedura, as fosfatases 2A desempenham um importante papel na via da quinase TOR, o que ocorre por meio da proteína essencial Tap42. A proteína Tip41 foi identificada como um parceiro de interação de Tap42 e regulador da via da quinase TOR em levedura. A homóloga de Tap42 em mamíferos, chamada de a4, está envolvida na regulação de diversos processos celulares, como diferenciação, desenvolvimento, migração celular e apoptose, por meio de seu papel conservado de regulador de fosfatases 2A. A homóloga em mamíferos de Tip41, chamada TIPRL, é uma proteína ainda pouco caracterizada. Este trabalho teve como objetivo analisar a função das proteínas a4 e TIPRL humanas e esclarecer seu papel na regulação de fosfatases 2A. A caracterização estrutural de a4 e Tap42, usando dados de SAXS, dicroísmo circular e proteólise limitada, mostrou que essas proteínas apresentam um domínio N-terminal compacto formado por a-hélices e um domínio C-terminal desestruturado. Em uma triagem de interações com a proteína TIPRL humana, identificamos as fosfatases PP2Ac, PP4c e PP6c como seus parceiros de interação, assim como os fatores de transcrição MafB e TAF10. Ao contrário do esperado a partir do modelo de levedura, a4 e TIPRL não interagem diretamente, mas formam um complexo ternário com PP2Ac. Uma triagem de substratos de fosfatases 2A regulador por TIPRL identificou os fatores de splicing SF2/ASF e SF2p32. Nossos resultados sugerem um modelo estrutural para a regulação das fosfatases 2A por a4 e mostram que TIPRL é um novo regulador comum dessas fosfatases com funções na regulação da expressão gênica.Abstract: Cells respond constantly to a variety of stimuli, which are interpreted and integrated through signaling networks, giving rise to biological responses. Defects in this circuitry are a cause of many diseases, including cancer. Protein phosphatases are enzymes which remove phosphate groups from kinase substrates, relying mainly on regulatory subunits for their substrate specificity. Type 2A phosphatases belong to the PPP subfamily, which is formed by PP2A, PP4 and PP6. PP2A is the major soluble serine/threonine phosphatase in animal cells and is found predominantly as a heterotrimer composed of a catalytic (C), a regulatory (B, B', B'' or B''') and a scaffold (PR65/A) subunit. Type 2A phosphatases play a major role in the yeast TOR signaling pathway through their interaction with the essential protein Tap42. Tip41 was identified as a Tap42 interacting protein and regulator of the TOR pathway. a4, the mammalian orthologue of Tap42, regulates many cellular processes such as differentiation, development, cell migration and apoptosis as a conserved type 2A phosphatase regulator. TIPRL, the mammalian orthologue of Tip41, is still poorly characterized. The objective of the present work was to analyse the function of a4 and TIPRL and improve the understanding of their role as type 2A phosphatase regulators. The structural characterization of a4 using SAXS analyses, circular dichroism and limited proteolysis, showed that these proteins are formed by an a-helical N-terminal domain and an unfolded C-terminal domain. A screen for TIPRL interacting proteins identified PP2Ac, PP4c and PP6c and also the transcription factors MafB and TAF10. Unlike their yeast conterparts, a4 and TIPRL do not interact directly, but rather form a ternary complex with PP2A. A search for type 2A phosphatase substrates regulated by TIPRL identified the splicing factor SF2/ASF and its regulatory protein SF2p32. Our results suggest a structural model for the regulation of type 2A phosphatases by a4 and show that TIPRL is a novel common regulator of these phosphatases which functions in regulation of gene expression.DoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

New Interaction Partners For Nek4.1 And Nek4.2 Isoforms: From The Dna Damage Response To Rna Splicing.

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins. This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.131

Crystal structure and regulation of the citrus pol III repressor MAF1 by auxin and phosphorylation

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrusThis work was supported by Sa˜ o Paulo Research Foundation (FAPESP grant 2011/20468-1). C.E.B. and A.F.Z.N. received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Peer reviewe

Human Nek7-interactor RGS2 is required for mitotic spindle organization

<p>The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.</p

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK613940744090CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Characterization of the Human NEK7 Interactome Suggests Catalytic and Regulatory Properties Distinct from Those of NEK6

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional <i>in vitro</i> and <i>in vivo</i> assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6