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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 133-208 and 133-208 Y149A F150A)

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kirk West

LAB HEAD: Justin W. Leung

PROVIDER: PXD048215 | Pride | 2025-01-02

REPOSITORIES: Pride

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			Action	DRS
	LeungJW_102422_133_204_S.raw	Raw
	LeungJW_102422_133_204_S.raw__F016892_.mzid.gz	Mzid
	LeungJW_102422_133_204_S.raw__F016892_.mzid_LeungJW_102422_133_204_S.raw__F016892_.MGF	Mzid
	LeungJW_102422_133_204_YF_S.raw	Raw
	LeungJW_102422_133_204_YF_S.raw__F016894_.mzid.gz	Mzid

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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 WT, 1-240, and D607A)

Project description: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.

2025-01-02 | PXD047964 | 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

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

SFB-tagged(S-protein tag, Flag epitope tag, and streptavidin-binding peptide tag) RNF4 wild-type and CS mutant LC-MSMS

Project description:To elucidate the role of RNF4 in DNA replication and fork reversal, we performed tandem affinity purification (TAP) using a HEK293T cell line that stably expresses SFB-tagged(S-protein tag, Flag epitope tag, and streptavidin-binding peptide tag) wild-type and CS mutant to isolate proteins that associate with RNF4.

2022-08-11 | PXD028397 | Pride

Transcription profiling of mouse reveals induction of intestinal Th17 cells by segmented filamentous bacteria

Project description:The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. The process by which the commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation, anti-microbial defenses, and tissue repair, and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Control of Th17 cell differentiation by SFB may thus establish a balance between optimal host defense preparedness and potentially damaging T cell responses. Manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease. Experiment Overall Design: We compared the gene expression profiles in the terminal ileum of Swiss-Webster GF mice before and after colonization with SFB, which induced robust Th17 cell differentiation. To sieve out host effects, the role of other microbiota, as well as other factors, we also evaluated the transcriptional program induced in Jackson C57BL/6 mice after co-housing with Taconic B6 animals, which also induces Th17 cell differentiation. 0.5 cm of the most distal part of the small intestine was dissected. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX10.

2009-11-10 | E-GEOD-18348 | biostudies-arrayexpress

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

Genome-wide distribution of UHRF2 and ZNF618 in 293T cells.

Project description:We have identified ZNF618 as a novel binding partner of UHRF2. We have found that UHRF2 and ZNF618 co-localize at many genomic loci. Examination of genome-wide distribution of SFB-tagged UHRF2 and ZNF618 in 293T cells using ChIP-seq.

2016-05-31 | E-GEOD-80037 | biostudies-arrayexpress

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

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