Dataset Information

Molecular basis of Tousled-Like-Kinase 2 (TLK2) activation – LC-MS/MS-based analysis of HEK293-expressed TLK2 phosphorylation state

ABSTRACT: In this study on human Tousled-Like-Kinase 2 (TLK2) mass spectrometry-based analysis was applied as part of a molecular characterization of TLK2 in the aim to increase the understanding of the mode of activation of this protein. Specifically, different TLK2 constructs were expressed analyzed to generate a “phosphorylation map” of the TLK2 protein. To analyse the phosphorylation state of TLK2 we used recombinant protein purified from HEK293. Samples of different TLK2 constructs were used for in-gel digestion and analysed by mass spectrometry. All samples were prepared and run in triplicates for label-free quantification. Explanation for MS raw files with TLK2 construct included: KD: Kinase-dead TLK2 construct with mutation D613A, N-terminal His- and Strep-tag. wt: Active form of TLK2, canonical sequence, N-terminal His- and Strep-tag. full: Full-length canonical sequence (1-772), N-terminal His- and Strep-tag. trunc: Truncated TLK2 sequence (191-772), N-terminal His- and Strep-tag.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Patrick Ruether

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD009095 | Pride | 2018-09-04

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	171020SwissProtHuman9606canonical.fasta	Fasta
	20180202_QE3_nLC11_PR_SA_TLK2_KD-full_1.raw	Raw
	20180202_QE3_nLC11_PR_SA_TLK2_KD-full_2.raw	Raw
	20180202_QE3_nLC11_PR_SA_TLK2_KD-full_3.raw	Raw
	20180202_QE3_nLC11_PR_SA_TLK2_KD-trunc_1.raw	Raw

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Publications

Molecular basis of Tousled-Like Kinase 2 activation.

Mortuza Gulnahar B GB Hermida Dario D Pedersen Anna-Kathrine AK Segura-Bayona Sandra S López-Méndez Blanca B Redondo Pilar P Rüther Patrick P Pozdnyakova Irina I Garrote Ana M AM Muñoz Inés G IG Villamor-Payà Marina M Jauset Cristina C Olsen Jesper V JV Stracker Travis H TH Montoya Guillermo G

Nature communications 20180628 1

Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domai ...[more]

PMID: 29955062

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Project description:As part of our study on human MASTL (microtubule-associated serine/threonine kinase-like), we used mass spectrometry (MS)-based proteomics in the aim to unravel novel aspects about MASTL activation, regulation and potential substrates. Specifically, we performed a phosphoproteomics-based kinase assay (essentially as in Xue et al. 2014) to identify MASTL substrates and analyzed different MASTL constructs to generate a “phosphorylation map” of the MASTL protein. siKALIP (Stable Isotope Labelled Kinase Assay-Linked Phosphoproteomics) Three MASTL variants, which we termed MASTL (full-length), MASTL450 (short) and Bonsai (truncated), were expressed in HEK293 cells and isolated using a tandem Strep and His-tag affinity purifications. For the in vitro kinase assay we used a dephosphorylated cell extract from interphase HEK293 cells to perform a slightly modified version of the siKALIP (described in Xue et al. 2014). Samples included an untreated basal sample, a dephosphorylated sample and samples for each of the three MASTL constructs. For label-free quantification, reactions were performed in quadruplicates for each MASTL construct. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length. 450short: Shorter (consisting of the first 450 amino acid residues) MASTL. Bonsai: Truncated MASTL (consisting of amino acid residues 35-112, 113-180 and 728-879). MASTL phosphorylation map To analyse the phosphorylation state of MASTL we used isolated protein from an interphase HEK293 cell culture, either alone or incubated with mitotic extracts from HEK293 cells. Samples of different MASTL constructs were used for in-gel digestion and analysed by MS. Explanation for MS raw files with MASTL construct included: FL: MASTL full-length (“INACTIVE” indicates kinase-dead, “Dephos” indicates dephosphorylated FL protein, “Mit” indicates incubated with mitotic extracts). 450: Shorter (450 amino acids) MASTL (“KD” indicates kinase-dead). BONSAI: Truncated MASTL (“KD” indicates kinase-dead).