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Autophosphorylation of Tousled-like kinases (TLK1 and TLK2) regulates recruitment to damaged chromatin via PCNA interaction (Tandem Affinity Purification coupled to LC-MS/MS of SFB-TLK1 WT, 1-240, and D607A)

ABSTRACT: Tandem affinity purification of SFB-Tagged TLK1 Wildtype, TLK1 1-240, and TLK1 D607A from transiently transfected HEK 293T cells to try to identify possible TLK1 interactions. Data represent technical duplicates.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kirk West

LAB HEAD: Justin W. Leung

PROVIDER: PXD047964 | Pride | 2025-01-02

REPOSITORIES: pride

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			Action	DRS
	Leung_071921_TLK1_KD_Chrom.raw	Raw
	Leung_071921_TLK1_KD_Chrom_2.raw	Raw
	Leung_071921_TLK1_KD_Sol.raw	Raw
	Leung_071921_TLK1_KD_Sol_2.raw	Raw
	Leung_071921_TLK1_Nterm_Chrom.raw	Raw

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1 - 5 of 18

Publications

Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction.

West Kirk L KL Nguyen Tram T N TTN Tengler Kyle A KA Kreiling Natasha N Raney Kevin D KD Ghosal Gargi G Leung Justin W JW

Nucleic acids research 20241227

Tousled-like kinases 1 and 2 (TLK1 and 2) are cell cycle-regulated serine/threonine kinases that are involved in multiple biological processes. Mutation of TLK1 and 2 confer neurodegenerative diseases. Recent studies demonstrate that TLK1 and 2 are involved in DNA repair. However, there is no direct evidence that TLK1 and 2 function at DNA damage sites. Here, we show that both TLK1 and TLK2 are hyper-autophosphorylated at their N-termini, at least in part, mediated by their homo- or hetero- dime ...[more]

PMID: 39727191

Similar Datasets

Autophosphorylation of Tousled-like kinases (TLK1 and TLK2) regulates recruitment to damaged chromatin via PCNA interaction (Tandem Affinity Purification coupled to LC-MS/MS of SFB-TLK1 133-208 and 133-208 Y149A F150A)

Project description:Tandem affinity purification of HEK 293T cells transiently expressing SFB-TLK1 133-208 or SFB-TLK1 133-208 Y149A F150A to identify if TLK1 PIP box mutants can still interact with PCNA.

2025-01-02 | PXD048215 | Pride

Autophosphorylation of Tousled-like kinases (TLK1 and TLK2) regulates recruitment to damaged chromatin via PCNA interaction (Phospho-TMT analysis of TLK1)

Project description:PhosphoTMT experiment on HEK293T cells transiently transfected with either SFB-TLK1, SFB-TLK2, or siRNAs targeting TLK1, TLK2, or TLK1 and TLK2. Data represents 4 biological replicates.

2025-01-02 | PXD047966 | Pride

Mutational analysis of TLK1 and MDM1

Project description:The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with ‘Intellectual Disability, Autosomal Dominant 57’ (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a heterozygous TLK1 variant (c.1435C>G, p.Q479E), as well as a mutation in MDM1 (c.1197dupT, p.K400*). Cells expressing TLK1 p.Q479E exhibited reduced cytokine responses and elevated DNA damage, but not increased radiation sensitivity or DNA repair defects. The TLK1 p.Q479E variant impaired kinase activity but not proximal protein interactions.

2024-04-30 | GSE266289 | GEO

Project description:S-protein-2xFLAG-Streptavidin binding peptide (SFB)-tagged TLK1 or SFB-TLK2 were transfected into 4x 15 cm plates of HEK293T cells. Cells were harvested 24 hours after transfection using NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% NP-40) supplemented with 2 µg/mL aprotinin (Thermo, AAJ60237MB) and 5 µg/mL pepstatin A (Thermo, PI78432) at 4ºC for 20 minutes. Lysates were centrifuged at 9,000 x g, 4ºC for 20 minutes to yield supernatant as soluble fraction. Soluble fractions were incubated with streptavidin sepharose (200 µl) (GE Healthcare, GE17-5113-01) at 4ºC for 1 hour followed by washing with NETN buffer three times. The protein complexes were eluted with 2 mg/mL biotin at 4ºC for 1 hour. The eluents were then incubated with S-protein agarose (EMD Millipore, 69704-3) overnight at 4ºC, washed three times with NETN buffer, and eluted in 1x Laemmli buffer.

2025-01-02 | PXD047996 | Pride

USP11 deubiquitinates monoubiquitinated SPRTN to repair DNA-protein crosslinks

Project description:DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. SPRTN is a replication-coupled DNA-dependent metalloprotease that degrades proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN chromatin accessibility during DPC repair. The deubiquitinase regulating SPRTN function in DPC repair is unknown. To identify SPRTN modifiers, we performed tandem-affinity purification and mass spectrometry (TAP-MS) analysis of SFB (S-FLAG-streptavidin binding peptide)-tagged SPRTN expressed in HEK 293T cells. We screened the MS list for proteins that are known to function as a deubiquitinase and identified only one potential SPRTN modifier, USP11 deubiquitinase. A reciprocal TAP-MS analysis of SFB-USP11 expressed in HEK 293T cells treated with camptothecin (CPT) immunoprecipitated SPRTN. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impaired SPRTN deubiquitination in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings elucidates the function of USP11 in the regulation of SPRTN monoubiquitination and SPRTN-mediated DPC repair.

2021-04-01 | PXD019923 | Pride

TLK1 phosphorylates human RAD54

Project description:TLK1 phosphorylates RAD54 at novel residues -T41, T59, T700

2023-10-24 | PXD037131 | Pride

De novo TLK1 and MDM1 mutations in a patient with a neurodevelopmental disorder and immunodeficiency

Project description:The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with ‘Intellectual Disability, Autosomal Dominant 57’ (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling.

2023-08-18 | GSE241032 | GEO

Mutational analysis of TLK1 and MDM1

Project description:Mutational analysis of TLK1 and MDM1

| PRJNA1106510 | ENA

Identification of dASF/SF2 potential mRNA targets

Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at identifying dASF/SF2 potential mRNA targets in the Drosophila developing eye Control: GMR X GFP-NLS trangenic larvae The 2 IP- GFP cy3 vs ASF cy5 rep.1 and IP- GFP cy5 vs ASF cy3 rep.1 samples correspond to dye-swap experiments. Two replicates (rep1 and rep2) are included.

2007-09-17 | E-GEOD-5857 | biostudies-arrayexpress

Identification of B52 potential mRNA targets

Project description:Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout growth of a whole organism. Targeting expression of five SR proteins in the developing eye of Drosophila allowed us to show that these splicing factors induce various phenotypic alterations concerning eye organogenesis and viability. Although both dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impairs normal photoreceptor axons projection and neurogenesis in visual ganglia. Consistently, microarray analyses revealed that many of the B52 targets are involved in brain organogenesis and we show that their splicing profile is altered both in B52 loss and gain of function. Conversely, a large proportion of dASF/SF2 targets are involved in eye development. This differential effect argues that SR proteins confer accuracy to developmental gene-expression programs, thus ensuring tissue identity and supporting cell-lineage decisions. Keywords: genetic modification Experiment aimed at identifying B52 potential mRNA targets in the Drosophila developing eye Control: GMR X GFP-NLS trangenic larvae The 2 IP- GFP cy3 vs B52 cy5 rep.1 and IP- GFP cy5 vs B52 cy3 rep.1 samples correspond to dye-swap experiments. Two replicates (rep1 and rep2) are included.

2007-09-17 | E-GEOD-5858 | biostudies-arrayexpress

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