Integrated Analysis of Global mRNA and Protein Expression Data in HEK293 Cells Overexpressing PRL-1

Dumaual, Carmen M.; Steere, Boyd; Walls, Chad D.; Wang, Mu; Zhang, Zhong Yin; Randall, Stephen K.

doi:10.1371/journal.pone.0072977

Cited by 11 publications

(5 citation statements)

References 119 publications

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“…To assess LCK autophosphorylation activity, we expressed full-length LCK tagged with Myc in HEK293 cells, which lack endogenous LCK (62), and monitored the production of the protein by Western blot with an antibody against the Myc epitope and the amount of phosphorylated LCK with an antibody recognizing LCK phosphorylated at Tyr 394 , which is in the activation loop (Fig. 4B).…”

Section: Resultsmentioning

confidence: 99%

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Malecka

Fink

et al. 2015

Sci. Signal.

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Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.

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Section: Resultsmentioning

confidence: 99%

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Malecka

Fink

et al. 2015

Sci. Signal.

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“…Finally, over-expression of eIF6 in primary melanoma cell lines (WM793) induced cdc42 up-regulation and increased motility and invasiveness, thus demonstrating that the tumor-promoting ability of this initiation factor is not restricted to the A2780 cell line. Notably, both eIF6 and cdc42 have been reported to be up-regulated in cells over-expressing PRL-1, a putative oncogene involved in the control of a number of diverse biological processes, including migration and invasion [ 31 ]. Our results could suggest a possible mechanism of regulation in which eIF6 act as a mediator of the cdc42 expression at the translational level, although additional experiments are needed to elucidate this issue.…”

Section: Discussionmentioning

confidence: 99%

eIF6 over-expression increases the motility and invasiveness of cancer cells by modulating the expression of a critical subset of membrane-bound proteins

et al. 2015

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BackgroundEukaryotic Initiation factor 6 (eIF6) is a peculiar translation initiation factor that binds to the large 60S ribosomal subunits, controlling translation initiation and participating in ribosome biogenesis. In the past, knowledge about the mechanisms adopted by the cells for controlling protein synthesis by extracellular stimuli has focused on two translation initiation factors (eIF4E and eIF2), however, recent data suggest eIF6 as a newcomer in the control of downstream of signal transduction pathways. eIF6 is over-expressed in tumors and its decreased expression renders cells less prone to tumor growth. A previous work from our laboratory has disclosed that over-expression of eIF6 in transformed cell lines markedly increased cell migration and invasion.MethodsHere, we performed a quantitative proteomic analysis of membrane-associated proteins in A2780 ovarian cancer cells over-expressing eIF6. Differentially expressed proteins upon eIF6 overproduction were further investigated in silico by Ingenuity Pathway Analysis (IPA). RT-qPCR and Western blot were performed in order to validate the proteomic data. Furthermore, the effects of a potent and selective inhibitor ML-141 in A2780 cells were evaluated using transwell migration assay. Finally, we explored the effects of eIF6 over-expression on WM793 primary melanoma cell lines.ResultsWe demonstrated that: (i) the genes up-regulated upon eIF6 overproduction mapped to a functional network corresponding to cellular movements in a highly significant way; (ii) cdc42 plays a pivotal role as an effector of enhanced migratory phenotype induced upon eIF6 over-expression; (iii) the variations in abundance observed for cdc42 protein occur at a post-transcriptional level; (iv) the increased cell migration/invasion upon eIF6 over-expression was generalizable to other cell line models.ConclusionsCollectively, our data confirm and further extend the role of eIF6 in enhancing cell migration/invasion. We show that a number of membrane-associated proteins indeed vary in abundance upon eIF6 over-expression, and that the up-regulated proteins can be located within a functional network controlling cell motility and tumor metastasis. Full understanding of the role eIF6 plays in the metastatic process is important, also in view of the fact that this factor is a potentially druggable target to be exploited for new anti-cancer therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1106-3) contains supplementary material, which is available to authorized users.

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“…Therefore, a comprehensive understanding on biological systems will require extending single omics further to multi‐omics analysis for gene therapy research. Multi‐omics studies have been conducted in other biologics research (Ang et al, 2019; Dumaual et al, 2013; Shin et al, 2018; Stolfa et al, 2018). Similarly, multi‐omics can be applied to understand the challenges and bottlenecks for rAAV production, such as issues in low productivity, scale‐up, and establishing stable producers.…”

Section: Advances In Gene Therapy Biomanufacturingmentioning

confidence: 99%

Critical challenges and advances in recombinant adeno‐associated virus (rAAV) biomanufacturing

Polanco

Lee

et al. 2023

Biotech & Bioengineering

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Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno‐associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV‐producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV‐mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state‐of‐the‐art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.

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Integrated Analysis of Global mRNA and Protein Expression Data in HEK293 Cells Overexpressing PRL-1

Cited by 11 publications

References 119 publications

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

eIF6 over-expression increases the motility and invasiveness of cancer cells by modulating the expression of a critical subset of membrane-bound proteins

Critical challenges and advances in recombinant adeno‐associated virus (rAAV) biomanufacturing

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