Dataset Information

In Cellulo Kinase Screen Identifies Novel MASTL Substrates

ABSTRACT: MASTL (microtubule-associated serine/threonine kinase-like) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. With the cellular roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are any additional substrates and if so, what the optimal consensus motif for MASTL is. Here we utilised a whole cell lysate (in cellulo) kinase screen approach combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify novel substrates and a consensus motif of MASTL. Using the related AGC kinase family members AKT1/2, the in cellulo assay identified several known and new substrates highly enriched with the validated consensus motif for AKT. Applying this method to MASTL identified 59 phosphosites on 26 novel proteins significantly increased in the presence of active MASTL. Subsequent in vitro kinase assays confirmed that MASTL was capable of phosphorylating YB1, RPS6, TUBA1C, and RPL36A under some conditions, and hnRNPM specifically on a single site (S-633/7). Taken together, these data suggest that MASTL can phosphorylate several additional substrates, which may help provide greater insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

INSTRUMENT(S): Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Kamila Marzec

LAB HEAD: Andrew Burgess

PROVIDER: PXD028678 | Pride | 2022-08-12

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	InCelluloKinaseScreenIdentifiesNovelMASTLSubstrates_PRIDEsubmission.xlsx	Xlsx
	RAW_AKT1_YB1.zip	Other
	RAW_MASTL_ENSA.zip	Other
	RAW_MASTL_ENSA_repeats.zip	Other
	RAW_MASTL_HNRNPM.zip	Other

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Publications

SILAC kinase screen identifies potential MASTL substrates.

Marzec Kamila A KA Rogers Samuel S McCloy Rachael R Parker Benjamin L BL James David E DE Watkins D Neil DN Burgess Andrew A

Scientific reports 20220622 1

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kin ...[more]

PMID: 35732702

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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).