Dyrk2-associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation

Telomerase maintains the telomere, a specialized chromosomal end structure that is essential for genomic stability and cell immortalization. Telomerase is not active in most somatic cells, but its reactivation is one of the hallmarks of cancer. In this study, we found that dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (Dyrk2) negatively regulates telomerase activity. Dyrk2 phosphorylates TERT protein, a catalytic subunit of telomerase. Phosphorylated TERT is then associated with the EDD-DDB1-VprBP E3 ligase complex for subsequent ubiquitin-mediated TERT protein degradation. During the cell cycle, Dyrk2 interacts with TERT at the G2/M phase and induces degradation. In contrast, depletion of endogenous Dyrk2 disrupts the cell cycle-dependent regulation of TERT and elicits the constitutive activation of telomerase. Similarly, a Dyrk2 nonsense mutation identified in breast cancer compromises ubiquitination-mediated TERT protein degradation. Our findings suggest the novel molecular mechanism of kinase-associated telomerase regulation.     

CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes

Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies.     

Regulation of cellular immortalization and steady-state levels of the telomerase reverse transcriptase through its carboxy-terminal domain

Telomerase maintains cell viability and chromosomal stability through the addition of telomere repeats to chromosome ends. The reactivation of telomerase through the upregulation of TERT, the telomerase protein subunit, is an important step during cancer development, yet TERT protein function and regulation remain incompletely understood. Despite its close sequence similarity to human TERT (hTERT), we find that mouse TERT (mTERT) does not immortalize primary human fibroblasts. Here we exploit these differences in activity to understand TERT protein function by creating chimeric mouse-human TERT proteins. Through the analysis of these chimeric TERT proteins, we find that sequences in the human carboxy-terminal domain are critical for telomere maintenance in human fibroblasts. The substitution of the human carboxy-terminal sequences into the mouse TERT protein is sufficient to confer immortalization and maintenance of telomere length and function. Strikingly, we find that hTERT protein accumulates to markedly higher levels than does mTERT protein and that the sequences governing this difference in protein regulation also reside in the carboxy-terminal domain. These elevated protein levels, which are characteristic of hTERT, are necessary but not sufficient for telomere maintenance because stabilized mTERT mutants cannot immortalize human cells. Thus, the TERT carboxy terminus contains sequences that regulate TERT protein levels and determinants that are required for productive action on telomere ends.