A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins

Merrill, Ronald A.; Song, Jianing; Kephart, Rikki A.; Klomp, Annette J.; Noack, Claire E.; Strack, Stefan

doi:10.1074/jbc.ra119.010596

Cited by 33 publications

(27 citation statements)

References 16 publications

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“…To investigate DCX protein turnover and its dependence on KLHL15 and the FRY sequence, we analyzed substrate protein degradation using a HaloTag-based pulse-chase protocol, which we recently developed for long-lived proteins ( 41 ). HEK293T cells expressing endogenous KLHL15 ( 15 ) were cotransfected with DCX-HaloTag fusion cDNAs (WT or Y259L) and either GFP-KLHL15 or KLHL15-directed shRNAs ( 15 ), followed by pulse-labeling with a cell-permeant and covalent HaloTag ligand coupled to the fluorophore tetramethyl rhodamine (TMR).…”

Section: Resultsmentioning

confidence: 99%

“…HEK293T cells expressing endogenous KLHL15 ( 15 ) were cotransfected with DCX-HaloTag fusion cDNAs (WT or Y259L) and either GFP-KLHL15 or KLHL15-directed shRNAs ( 15 ), followed by pulse-labeling with a cell-permeant and covalent HaloTag ligand coupled to the fluorophore tetramethyl rhodamine (TMR). Labeled DCX-HaloTag was chased by incubating cells with the non-fluorescent competitive ligand 7-bromoheptanol for up to 24 h ( 41 ). DCX turnover was quantified by dividing TMR fluorescence by DCX immunoblot signals in the same lane and normalizing to the zero time point.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were grown to 60% confluency on collagen-coated plates and transfected using Lipofectamine 2000 (Invitrogen) following the manufacture's protocol for transient transfection of adhered cells. Primary hippocampal cultures were prepared from embryonic day 18 (E18) Sprague Dawley rats (Harlan) as described previously ( 41 ). Hippocampi were dissected and pooled in ice-cold HEPES-buffered neurobasal and then incubated in HBSS containing trypsin (1.5 mg/ml) at 37 °C for 12 min.…”

Section: Methodsmentioning

confidence: 99%

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The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

Song¹,

Merrill²,

Usachev

et al. 2021

Journal of Biological Chemistry

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Proper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability (XLID). Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

Song¹,

Merrill²,

Usachev

et al. 2021

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…To investigate DCX protein turnover and its dependence on KLHL15 and the FRY sequence, we analyzed substrate protein degradation using a HaloTag-based pulsechase protocol, which we recently developed for long-lived proteins (41). HEK293T cells expressing endogenous KLHL15 (15) were cotransfected with DCX-HaloTag fusion cDNAs (wild-type or Y259L) and either GFP-KLHL15 or KLHL15-directed shRNAs (15), followed by pulse-labeling with a cellpermeant and covalent HaloTag ligand coupled to the fluorophore tetramethyl rhodamine (TMR).…”

Section: Tyr Within the Fry Sequence Of DCX Proteins Is Critical For mentioning

confidence: 99%

“…Medium was changed to serum-free Neurobasal complete after 4 h of incubation. Cells were maintained at 37 °C in a humidified environment of 95% air/5% CO2 with half the media changed with fresh media twice a week (41).…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

Song

Merrill

Usachev

et al. 2020

Preprint

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Proper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability. Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease gene, and doublecortin-like kinases 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

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A fluorescence‐based sensor for calibrated measurement of protein kinase stability in live cells

Paul,

Muratcioğlu,

Kuriyan

2024

Protein Science

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Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein‐kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co‐expressed reference fluorescent proteins. We used this tool to study the dependence of Src‐ and Raf‐family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src‐homology 2 and Src‐homology 3 domains, which are required for autoinhibition of Src‐family kinases, stabilize these kinase domains in the cell. Our expression‐calibrated sensor enables the facile characterization of the effects of mutations and small‐molecule drugs on protein‐kinase stability.

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A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins

Cited by 33 publications

References 16 publications

The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

A fluorescence‐based sensor for calibrated measurement of protein kinase stability in live cells

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