Purification, auto-activation and kinetic characterization of apoptosis signal-regulating kinase I

Pleinis, John M.; Davis, Cameron W.; Cantrell, Caleb B.; Qiu, David Y.; Zhan, Xiaoli

doi:10.1016/j.pep.2017.01.002

Cited by 7 publications

(8 citation statements)

References 49 publications

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“…Arrestin-3 (1-393) was expressed and purified as described (91)(92)(93)(94)(95) followed by an additional purification step on a Superdex S200 Increase 10/300 GL column (GE Life Sciences, Pittsburgh, PA) equilibrated in 20 mM MOPS, pH 7.5, 150 mM NaCl, and 2 mM TCEP. Protein purity was assessed by SDS-PAGE with Coomassie staining.…”

Section: Arrestin-3-(1-393) Expression and Purificationmentioning

confidence: 99%

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Kaya

Perry

Gurevich

et al. 2020

Preprint

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Agonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gas, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gas coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed. Significance Statement:The functional importance of receptor phosphorylation in GPCR regulation has been demonstrated. Over the past decade, the phospho-barcode concept was developed to explain the multidimensional nature of the arrestin-dependent signaling network downstream of GPCRs. Here, we used the dopamine-1 receptor (D1R) to explore the effect of receptor phosphorylation on G protein-dependent and arrestin-dependent ERK and Src activation. Our studies suggest that D1R intracellular loop-3 phosphorylation affects both G proteins and arrestins. Differential D1R phosphorylation can direct signaling toward ERK or Src activation. This implies that phosphorylation induces different conformations of receptor and/or bound arrestin to initiate or select different cellular signaling pathways. \body Although it is tempting to divide GPCR signaling into these two branches, G protein-dependent and arrestindependent, the nature of signaling in the cell is much more complex (reviewed in (1, 7, 8)). As a result, this classical paradigm does not fully explain the range of biological responses observed when a GPCR is activated. For example, agonist identity can affect the recruitment of different G-proteins or arrestins, which determines the selection of downstream effectors (9). In addition, some activated GPCRs may interact directly with non-canonical signaling partners, including Src-family kinases (10-21). There are also numerous studies suggesting a specific role for the nonvisual arrestins in receptor-independent activation of ERK1/2, AKT, JNK3, and NF-kB (6,(22...

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Section: Arrestin-3-(1-393) Expression and Purificationmentioning

confidence: 99%

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Kaya

Perry

Gurevich

et al. 2020

Preprint

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“…It is important to note that in vivo the main activators of MAPK cascades are not GPCRs. In most cases upstream MAP3Ks are activated by growth factor receptors[19,59], death receptors[60], integrins[61], or various stressors[62]. We should keep in mind that experimental paradigms used to study arrestin-mediated signaling actually exclude non-GPCR inputs.…”

Section: Mapks Are Activated By Various Inputsmentioning

confidence: 99%

“…Another example of complex activation mechanism is ASK1, one of the MAP3Ks of JNK1/2/3 cascades. It is activated by oxidative stress, endoplasmic reticulum (ER) stress, calcium influx, or mechanical stress, and inhibited by the interactions with reduced thioredoxin and 14-3-3 protein[62]. Its phosphorylation on three different serines in the N- and C-terminal elements is inhibitory, whereas the phosphorylation of the three threonines in the kinase domain is stimulatory[62].…”

Section: Mapks Are Activated By Various Inputsmentioning

confidence: 99%

“…It is activated by oxidative stress, endoplasmic reticulum (ER) stress, calcium influx, or mechanical stress, and inhibited by the interactions with reduced thioredoxin and 14-3-3 protein[62]. Its phosphorylation on three different serines in the N- and C-terminal elements is inhibitory, whereas the phosphorylation of the three threonines in the kinase domain is stimulatory[62]. Thus, in addition to being phosphorylated on threonines, for full activation the three serines in ASK1 must be dephosphorylated, and both thioredoxin and 14-3-3 protein must dissociate[62].…”

Section: Mapks Are Activated By Various Inputsmentioning

confidence: 99%

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Arrestin-mediated signaling: Is there a controversy?

Gurevich¹,

Gurevich²

2018

WJBC

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The activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK)1/2 was traditionally used as a readout of signaling of G protein-coupled receptors (GPCRs) via arrestins, as opposed to conventional GPCR signaling via G proteins. Several recent studies using HEK293 cells where all G proteins were genetically ablated or inactivated, or both non-visual arrestins were knocked out, demonstrated that ERK1/2 phosphorylation requires G protein activity, but does not necessarily require the presence of non-visual arrestins. This appears to contradict the prevailing paradigm. Here we discuss these results along with the recent data on gene edited cells and arrestin-mediated signaling. We suggest that there is no real controversy. G proteins might be involved in the activation of the upstream-most MAP3Ks, although in vivo most MAP3K activation is independent of heterotrimeric G proteins, being initiated by receptor tyrosine kinases and/or integrins. As far as MAP kinases are concerned, the best-established role of arrestins is scaffolding of the three-tiered cascades (MAP3K-MAP2K-MAPK). Thus, it seems likely that arrestins, GPCR-bound and free, facilitate the propagation of signals in these cascades, whereas signal initiation via MAP3K activation may be independent of arrestins. Different MAP3Ks are activated by various inputs, some of which are mediated by G proteins, particularly in cell culture, where we artificially prevent signaling by receptor tyrosine kinases and integrins, thereby favoring GPCR-induced signaling. Thus, there is no reason to change the paradigm: Arrestins and G proteins play distinct non-overlapping roles in cell signaling.

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“…ASK1 is a family member of the mitogenactivated protein kinase kinase kinase (MAP3K) 21 , acting as a signaling node in which different stressors such as endoplasmic reticulum, oxidative and inflammatory stresses converge 4 . In addition to being activated by different stressors, ASK1 signaling can be induced via autophosphorylation 22 suggesting that a mere increase in ASK1 expression results in elevated ASK1 activity. Importantly, elevated ASK1 expression in adipose tissue of obese human subjects was found to be an independent predictor of whole-body insulin resistance 23 , underscoring the potential importance of this stress-signaling pathway in obesity and associated metabolic disorders.…”

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confidence: 99%

ASK1 inhibits browning of white adipose tissue in obesity

et al. 2020

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Increasing energy expenditure via induction of adipose tissue browning has become an appealing strategy to treat obesity and associated metabolic complications. Herein, we identify adipocyte-expressed apoptosis signal-regulating kinase 1 (ASK1) as regulator of adipose tissue browning. High fat diet-fed adipocyte-specific ASK1 knockout mice reveal increased UCP1 protein levels in inguinal adipose tissue concomitant with elevated energy expenditure, reduced obesity and ameliorated glucose tolerance compared to control littermates. In addition, ASK1-depletion blunts LPS-mediated downregulation of isoproterenolinduced UCP1 in subcutaneous fat both in vitro and in vivo. Conversely, adipocyte-specific ASK1 overexpression in chow-fed mice attenuates cold-induced UCP1 protein levels in inguinal fat. Mechanistically, ASK1 phosphorylates interferon regulatory factor 3 (IRF3) resulting in reduced Ucp1 expression. Taken together, our studies unravel a role of ASK1 in mediating the inhibitory effect of caloric surplus or LPS-treatment on adipose tissue browning. Adipocyte ASK1 might be a pharmacological target to combat obesity and associated morbidities.

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Purification, auto-activation and kinetic characterization of apoptosis signal-regulating kinase I

Cited by 7 publications

References 49 publications

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Phosphorylation barcode-dependent signal bias of the dopamine D1 receptor

Arrestin-mediated signaling: Is there a controversy?

ASK1 inhibits browning of white adipose tissue in obesity

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