Roberto Magliozzi

Coupled activation and degradation of eEF2K regulates protein synthesis in response to genotoxic stress.

DNA damage triggers the phosphorylation of factors involved in protein synthesis to regulate polypeptide elongation. Activated, Then Degraded, Stop-Start Regulation of Protein Synthesis A key step in the process of translating mRNA into protein is the repositioning of the mRNA in the ribosome to enable elongation of the polypeptide chain. mRNA translocation in the ribosome is mediated by eukaryotic elongation factor 2 (eEF2), which is inhibited when phosphorylated by eEF2 kinase (eEF2K). Because protein synthesis is energetically costly, stressed cells inhibit this process to devote resources to stress responses. Kruiswijk et al. investigated the mechanisms by which genotoxic stress results in inhibition of protein synthesis. In response to a DNA-damaging agent, eEF2K was phosphorylated and activated by the kinase AMPK, thus leading to inhibition of eEF2 and a slowdown in elongation translation. eEF2K subsequently autophosphorylated itself in a motif recognized by the E3 ubiquitin ligase SCFβTrCP, and the resulting degradation of eEF2K enabled elongation translation to resume. Thus, the activation and subsequent degradation of eEF2K by genotoxic stress are coupled to inhibition of protein synthesis in response to DNA damage and resumption of protein synthesis after DNA damage has been resolved. The kinase eEF2K [eukaryotic elongation factor 2 (eEF2) kinase] controls the rate of peptide chain elongation by phosphorylating eEF2, the protein that mediates the movement of the ribosome along the mRNA by promoting translocation of the transfer RNA from the A to the P site in the ribosome. eEF2K-mediated phosphorylation of eEF2 on threonine 56 (Thr56) decreases its affinity for the ribosome, thereby inhibiting elongation. Here, we show that in response to genotoxic stress, eEF2K was activated by AMPK (adenosine monophosphate–activated protein kinase)–mediated phosphorylation on serine 398. Activated eEF2K phosphorylated eEF2 and induced a temporary ribosomal slowdown at the stage of elongation. Subsequently, during DNA damage checkpoint silencing, a process required to allow cell cycle reentry, eEF2K was degraded by the ubiquitin-proteasome system through the ubiquitin ligase SCFβTrCP (Skp1–Cul1–F-box protein, β-transducin repeat–containing protein) to enable rapid resumption of translation elongation. This event required autophosphorylation of eEF2K on a canonical βTrCP-binding domain. The inability to degrade eEF2K during checkpoint silencing caused sustained phosphorylation of eEF2 on Thr56 and delayed the resumption of translation elongation. Our study therefore establishes a link between DNA damage signaling and translation elongation.

Unraveling the ubiquitin-regulated signaling networks by mass spectrometry-based proteomics

Ubiquitin (Ub) is a small protein modifier that is covalently attached to the ε‐amino group of lysine residues of protein substrates, generally targeting them for degradation. Due to the emergence of specific anti‐diglycine (‐GG) antibodies and the improvement in MS, it is now possible to identify more than 10 000 ubiquitylated sites in a single proteomics study. Besides cataloging ubiquitylated sites, it is equally important to unravel the biological relationship between ubiquitylated substrates and the ubiquitin conjugation machinery. Relevant to this, we discuss the role of affinity purification‐MS (AP‐MS), in characterizing E3 ligase‐substrate complexes. Recently, such strategies have also been adapted to screen for binding partners of both deubiquitylating enzymes (DUBs) and ubiquitin‐binding domains (UBDs). The complexity of the “ubiquitome” is further expanded by the fact that Ub itself can be ubiquitylated at any of its seven lysine residues forming polyubiquitin (polyUb), thus diversifying its lengths and topologies to suit a variety of molecular recognition processes. Therefore, applying MS to study polyUb linkages is also becoming an emerging and important area. Finally, we discuss the future of MS‐based proteomics in answering important questions with respect to ubiquitylation.

Control of Epithelial Cell Migration and Invasion by the IKKβ- and CK1α-Mediated Degradation of RAPGEF2

Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-β and casein kinase-1α and consequently degraded by the proteasome via the SCF(βTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKβ and cell motility controlled by Rap-integrin signaling.

Proteasome-dependent degradation of transcription factor activating enhancer-binding protein 4 (TFAP4) controls mitotic division

Background: TFAP4 is a transcription factor that controls cell proliferation, stemness and epithelial-mesenchymal transition and is up-regulated in colorectal cancer. Results: TFAP4 is targeted for degradation by the SCFβTrCP ubiquitin ligase. Failure to degrade TFAP4 leads to aberrant mitosis. Conclusion: TFAP4 degradation is required for the fidelity of mitosis. Significance: Misregulation of TFAP4 might contribute to genomic instability and tumorigenesis. TFAP4, a basic helix-loop-helix transcription factor that regulates the expression of a multitude of genes involved in the regulation of cellular proliferation, stemness, and epithelial-mesenchymal transition, is up-regulated in colorectal cancer and a number of other human malignancies. We have found that, during the G2 phase of the cell division cycle, TFAP4 is targeted for proteasome-dependent degradation by the SCFβTrCP ubiquitin ligase. This event requires phosphorylation of TFAP4 on a conserved degron. Expression of a stable TFAP4 mutant unable to interact with βTrCP results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which eventually lead to the activation of the DNA damage response. Our findings reveal that βTrCP-dependent degradation of TFAP4 is required for the fidelity of mitotic division.

Degradation of Tiam1 by casein kinase 1 and the SCFβTrCP ubiquitin ligase controls the duration of mTOR-S6K signaling.

Background: The guanine nucleotide exchange factor Tiam1 regulates the activity of the small GTPase Rac1, a crucial regulator of cell adhesion, proliferation, and survival. Results: The SCFβTrCP ubiquitin ligase in cooperation with CK1 targets Tiam1 for proteasome-dependent degradation. Conclusion: Tiam1 degradation is required to terminate the mTOR-S6K signaling pathway. Significance: Tiam1 degradation controls the duration of mTOR-S6K signaling in response to mitogens. Tiam1 (T-cell lymphoma invasion and metastasis 1) is a guanine nucleotide exchange factor that specifically controls the activity of the small GTPase Rac, a key regulator of cell adhesion, proliferation, and survival. Here, we report that in response to mitogens, Tiam1 is degraded by the ubiquitin-proteasome system via the SCFβTrCP ubiquitin ligase. Mitogenic stimulation triggers the binding of Tiam1 to the F-box protein βTrCP via its degron sequence and subsequent Tiam1 ubiquitylation and proteasomal degradation. The proteolysis of Tiam1 is prevented by βTrCP silencing, inhibition of CK1 and MEK, or mutation of the Tiam1 degron site. Expression of a stable Tiam1 mutant that is unable to interact with βTrCP results in sustained activation of the mTOR/S6K signaling and increased apoptotic cell death. We propose that the SCFβTrCP-mediated degradation of Tiam1 controls the duration of the mTOR-S6K signaling pathway in response to mitogenic stimuli.

USP17- and SCFβTrCP-regulated degradation of DEC1 controls the DNA damage response

ABSTRACT In response to genotoxic stress, DNA damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the basic helix-loop-helix (bHLH) transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCFβTrCP ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1, markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through βTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response.

Nodal Signaling Range Is Regulated by Proprotein Convertase-Mediated Maturation

Tissue patterning is established by extracellular growth factors or morphogens. Although different theoretical models explaining specific patterns have been proposed, our understanding of tissue pattern establishment in vivo remains limited. In many animal species, left-right patterning is governed by a reaction-diffusion system relying on the different diffusivity of an activator, Nodal, and an inhibitor, Lefty. In a genetic screen, we identified a zebrafish loss-of-function mutant for the proprotein convertase FurinA. Embryological and biochemical experiments demonstrate that cleavage of the Nodal-related Spaw proprotein into a mature form by FurinA is required for Spaw gradient formation and activation of Nodal signaling. We demonstrate that FurinA is required cell-autonomously for the long-range signaling activity of Spaw and no other Nodal-related factors. Combined in silico and in vivo approaches support a model in which FurinA controls the signaling range of Spaw by cleaving its proprotein into a mature, extracellular form, consequently regulating left-right patterning.

Datasets from an interaction proteomics screen for substrates of the SCF(βTrCP) ubiquitin ligase

An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882.