Molecular basis of Tousled-Like Kinase 2 activation

Mortuza, Gulnahar B.; Hermida, Dario; Pedersen, Anna-Kathrine; Segura-Bayona, Sandra; López-Méndez, Blanca; Redondo, Pilar Negrete; Rüther, Patrick; Pozdnyakova, Irina; Garrote, Ana M.; Muñoz, Inés G.; Villamor-Payà, Marina; Jauset, Cristina; Olsen, Jesper V.; Stracker, Travis H.; Montoya, Guillermo

doi:10.1038/s41467-018-04941-y

Cited by 35 publications

(71 citation statements)

References 74 publications

(103 reference statements)

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“…A model of the TLK1B kinase domain was constructed by homology modeling using the ROBETTA de novo protein structure prediction server ( Song et al., 2013 ) (see Transparent Methods in Supplemental Information ) and further compared/aligned to the published crystal structure of the highly homologous TLK2 ( Mortuza et al., 2018 ). A PDB file for the structure was included as Data S1 .…”

Section: Resultsmentioning

confidence: 99%

“…To confirm these results and the specificity of the TLK1 inhibition with a genetic approach that could obviate potential off-target effects of J54, one possibility would be to deplete TLK1, but this is problematic because of the redundant function of TLK2. However, an alternative is to overexpress a dominant kinase-dead TLK1 (TLK1-KD) because this protein can inhibit both TLK1 and TLK2 as these kinases homo- and hetero-dimerize for full activation ( Mortuza et al., 2018 ). In fact, we have reported that overexpression of TLK1-KD efficiently suppresses pNek1-T141 ( Singh et al., 2017 ).…”

Section: Resultsmentioning

confidence: 99%

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Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

et al. 2020

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Summary Through in vitro kinase assays and docking studies, we report the synthesis and biological evaluation of a phenothiazine analog J54 with potent TLK1 inhibitory activity for prostate cancer (PCa) therapy. Most PCa deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa. Treatments that can suppress the conversion to mCRPC have high potential to be rapidly implemented in the clinics. ADT results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DNA damage response that typically results in temporary cell-cycle arrest of androgen-responsive PCa cells, whereas its abrogation leads to apoptosis. We studied J54 as a potent inhibitor of this axis and as a mediator of apoptosis in vitro and in LNCaP xenografts, which has potential for clinical investigation in combination with ADT. J54 has low affinity for the dopamine receptor in modeling and competition studies and weak detrimental behavioral effects in mice and C. elegans.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

et al. 2020

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“…In order to comprehensively map phosphorylation sites and to determine their stoichiometry in a purified protein by LC-MS/MS, it is essential to obtain its full sequence coverage 57 The mass spectrometric analysis of the ProAlanase digestion mixture resulted in 90% sequence coverage of the N3CID protein, while tryptic peptides covered 85% of the sequence (Fig. 5, a).…”

Section: Proline-rich Proteinmentioning

confidence: 99%

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

Samodova

Hosfield

Cramer

et al. 2020

Molecular & Cellular Proteomics

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Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in monoclonal antibody. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C-terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of non-collagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a non-reduced monoclonal antibody.

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“…Our variant is located at the N-terminus in a region where no functional domains are known ( Figure 3 ). This region however is expected to contain a nuclear localization signal (NLS) as mutants lacking the first 160 amino acids fail to localise to the nucleus ( 6 ). In addition, more than 20 phosphorylation sites, including p.Thr52 and p.Tyr70, have been identified in the N-terminal domain of TLK2, suggesting that this is potentially an important regulatory domain in vivo ( https://www.phosphosite.org ).…”

Section: Discussionmentioning

confidence: 99%

Severe neurodevelopmental disease caused by a homozygous TLK2 variant

et al. 2019

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A distinct neurodevelopmental phenotype characterised mainly by mild motor and language delay and facial dysmorphism, caused by heterozygous de novo or dominant variants in the TLK2 gene has recently been described. All cases reported carried either truncating variants located throughout the gene, or missense changes principally located at the C-terminal end of the protein mostly resulting in haploinsufficiency of TLK2. Through whole exome sequencing, we identified a homozygous missense variant in TLK2 in a patient showing more severe symptoms than those previously described, including cerebellar vermis hypoplasia and West syndrome. Both parents are heterozygous for the variant and clinically unaffected highlighting that recessive variants in TLK2 can also be disease causing and may act through a different pathomechanism.

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Molecular basis of Tousled-Like Kinase 2 activation

Cited by 35 publications

References 74 publications

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

Generation of Phenothiazine with Potent Anti-TLK1 Activity for Prostate Cancer Therapy

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

Severe neurodevelopmental disease caused by a homozygous TLK2 variant

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