Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKβ

Bustamante, Maria Blaire; Ansaloni, Annalisa; Pedersen, Jeppe Falsig; Azzollini, Lucia; Cariulo, Cristina; Wang, Zhe Ming; Petricca, Lara; Verani, Margherita; Puglisi, Francesca; Park, Hyunsun; Lashuel, Hilal A.; Caricasole, Andrea

doi:10.1016/j.bbrc.2015.06.116

Cited by 23 publications

(60 citation statements)

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“…Exon 1 of HTT also appears to assist in targeting huntingtin to autophagosomes and regulating HTT clearance by the lysosomal and proteasome pathways394041. In future work, it would be interesting to evaluate the impact of HTT N17 phosphorylation on the type of autophagy modulated by CCT (for example, selective versus bulk autophagy), as CCT and HTT may work together in autophagy and htt phosphorylation appears to modify its interactions395253. This might be important as WT huntingtin also functions as scaffold for selective autophagy4454.…”

Section: Discussionmentioning

confidence: 99%

“…As we used GFP-tagged constructs in most of our experiments, GFP tags may influence the degradation route options available to such proteins, for instance by increasing oligomerization and shifting flux from the proteasome to autophagy or by influencing phosphorylation events in htt that influence its clearance525657. However, it is important to point out that the clearance of these GFP-fusions are autophagy-dependent as are the clearance of untagged mutant htt58, mutant ataxin 3 (ref.…”

Section: Discussionmentioning

confidence: 99%

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CCT complex restricts neuropathogenic protein aggregation via autophagy

et al. 2016

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Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CCT complex restricts neuropathogenic protein aggregation via autophagy

et al. 2016

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“…Threonine 3 (T3) has been identified as one of the most common phosphorylation sites in Htt from ST14A and HeLa cells and was detected at lower levels in the post-mortem brain regions most affected by HD in a mouse model of HD (CAG140). [4,5] Interestingly, the level of T3 phosphorylation was inversely correlated with polyQ repeat length, suggesting that phosphorylation at T3 is decreased in pathological conditions and that restoring the physiological level of T3 phosphorylation might ameliorate HD pathology. [4] Testing this hypothesis has not been possible because the enzymes involved in regulating T3 phosphorylation remain elusive and the extent to which phosphomimetic mutations can reproduce the effect of phosphorylation remains unknown.…”

Section: Main Textmentioning

confidence: 99%

Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation‐Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6

et al. 2017

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Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 posttranslational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs. Main textSeveral lines of evidence suggest that the first N-terminal 17 amino acids of the Huntingtin protein (Htt) play important roles in modulating Htt structure, oligomerization, fibrils formation, membrane binding, subcellular localization and interactions with other proteins. [1] This, combined with the fact that this short N-terminal sequence harbors several residues that are subjected to diverse posttranslational modifications (PTMs), including phosphorylation, acetylation, ubiquitination and sumoylation (Scheme S1-A in the Supporting Information), suggests that PTMs may serve as reversible molecular switches for regulating Htt structure, interactome, cellular properties and toxicity. [2] However, the lack of knowledge about the enzymes involved in regulating these modifications has made it difficult to decipher their role in regulating the function(s) of Htt in health and disease.To address these limitations and enable deciphering of the Nt17 PTM code, our group has pursued the development of semisynthetic strategies that permit site-specific introduction of single or multiple PTMs within Nt17 of Huntingtin exon 1 (Httex1). Recently, we reported a semisynthetic strategy that permits site-specific phosphorylation at T3.[3] However, this strategy suffered from several limitations and was only suitable for introducing PTMs in the first N-terminal 9 residues of wild-type Httex1 (≤23 glutamine residues) (Scheme S2). [3] To allow investigations of all Nt17 PTMs, we developed and optimized two new modular semisynthetic protocols that permit the generation of wild-type and for the first time mutant Httex1 (mHttex1) proteins containing single or multiple PTMs (Scheme S3). The first strategy (Scheme S3-A) is only suitable for introducing PTMs in the N-terminal 9 residues of Httex1, whereas the second strategy allows the investigation of all the PTMs within the Nt17 domain. Additionally, the optimized conditions enabled the semisynthesis of mutant Httex1 (full details about these semisynthetic strategies are given in the Supporting Information). This optimized strategy enabled us, for...

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“…Mn 2ϩ -Phos-tag SDS-PAGE has been adapted to the analysis of phosphorylation of several proteins, e.g., ␣and ␤-caseins, ovalbumin (27), T cell antigen receptor subunits (11), the endoplasmic reticulum membrane proteins inositol-requiring enzyme-1␣ and protein kinase R-like endoplasmic reticulum kinase (63), CPI-17 (13,14,21), prostate-apoptosis response-4 (33), extracellular signal-regulated kinase 5 (43), hybrid sensor kinases (31), and huntingtin fragments (6). We have found it necessary to adjust various parameters to optimize the separation of the different phosphorylated species for each protein of interest.…”

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confidence: 99%

Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE

Sutherland

MacDonald

Walsh

2016

American Journal of Physiology-Cell Physiology

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Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.

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Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKβ

Cited by 23 publications

References 28 publications

CCT complex restricts neuropathogenic protein aggregation via autophagy

CCT complex restricts neuropathogenic protein aggregation via autophagy

Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation‐Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6

Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE

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