Profiling of p5, a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator, p35 CDKR1 Against 70 Protein Kinases

Binukumar, B.; Pelech, Steven; Sutter, Catherine; Shukla, Vibha; Amin, Niranjana D.; Grant, Philip; Bhaskar, Emmanuel; Skuntz, Susan; Steiner, Joseph P.; Pant, Harish C.

doi:10.3233/jad-160458

Cited by 6 publications

(5 citation statements)

References 37 publications

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“…Identification of TBK1 as a kinase that efficiently phosphorylates HTT at S13 in cellular models of HD To discover kinases responsible for phosphorylating HTT at S13 and S16, we used a commercial in vitro kinase screening assay (Kinexus in vitro kinase and phospho-peptide testing (IKPT) services), which includes~300 kinases (Binukumar et al, 2016). The screen was carried out using two HTT substrates: a peptide that comprises the first 17 N-terminal residues of HTT (N17), and the first exon of the HTT protein (HTTex1) ( Fig 1A-C).…”

Section: Resultsmentioning

confidence: 99%

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

et al. 2020

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Phosphorylation of the N‐terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK‐binding kinase 1 (TBK1), that efficiently phosphorylates full‐length and N‐terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1‐mediated neuroprotective effects are due to phosphorylation‐dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.

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Section: Resultsmentioning

confidence: 99%

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

et al. 2020

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“…Identification of TBK1 as a kinase that efficiently phosphorylates HTT at S13 in cellular models of HD To discover kinases responsible for phosphorylating HTT at S13 and S16, we used a commercial in vitro kinase screening assay, which includes ~300 kinases (Binukumar et al, 2016), using two HTT substrates; a peptide that comprises the first 17 N-terminal residues of HTT (N17), and the first exon of the HTT protein (HTTex1) ( Fig. 1A-C).…”

Section: Resultsmentioning

confidence: 99%

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Hegde

Chiki

Petricca³

et al. 2019

Preprint

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Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntington's Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD.3

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“…To enable the site‐specific enzymatic phosphorylation of Httex1 at T3, S13 or S16, we first needed to identify enzymes that phosphorylate HTT at these sites. Towards this goal, we performed a screening using the in vitro kinase and phosphopeptide testing (IKPT) services from Kinexus, [27] where Httex1‐23Q and the Nt17 peptide were used as substrates to test a panel of 298 purified serine‐threonine kinases. This led to the identification of several enzymes that phosphorylate Nt17 and Httex1 at both S13 and S16 (TBK1) or at T3 (GCK, TNIK, and HGK), respectively.…”

Section: Resultsmentioning

confidence: 99%

Site‐Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases

et al. 2020

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Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild-type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK), and 2) the development of an efficient methodology for producing recombinant native Httex1 proteins by using a SUMO-fusion expression and purification strategy. [26] As a proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site-specifically phosphorylated and fluorescently or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 or HTT structure, aggregation, interactome, and function(s) in health and disease.

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Profiling of p5, a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator, p35 CDKR1 Against 70 Protein Kinases

Cited by 6 publications

References 37 publications

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

Site‐Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases

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