Assay of protein kinases using radiolabeled ATP: a protocol

Hastie, C. James; McLauchlan, Hilary; Cohen, Philip

doi:10.1038/nprot.2006.149

Cited by 222 publications

(203 citation statements)

References 11 publications

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“…We also tested the ability of PAWS1 to influence CK1α activity against a peptide substrate (CK1tide) 33. First, we detected robust CK1α kinase activity in endogenous PAWS1 IPs from wild‐type but not PAWS1 −/− U2OS cell extracts (Fig 6A).…”

Section: Resultsmentioning

confidence: 98%

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PAWS 1 controls Wnt signalling through association with casein kinase 1α

Bozatzi

Dingwell

et al. 2018

EMBO Reports

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The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.

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Section: Resultsmentioning

confidence: 98%

“…Radioactivity was analysed by autoradiography. Kinase assays against substrate peptide (CK1tide, DSTT EP5630) were performed as described previously 33.…”

Section: Methodsmentioning

confidence: 99%

PAWS 1 controls Wnt signalling through association with casein kinase 1α

Bozatzi

Dingwell

et al. 2018

EMBO Reports

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“…Radioactive kinase reactions (typically 35 µl) were carried out with recombinant highly purified proteins, using a standard protocol similarly as described 60 . P-TEFb (0.1 µM) was pre-incubated with GST-CTD (100 µM) of multiple repeats and Hexim proteins for 10 min at room temperature in kinase buffer (50 mM HEPES, pH 7.6, 34 mM KCl, 7 mM MgCl 2 , 2.5 mM dithiothreitol, 5 mM β-glycerol phosphate, 0.5 mM Na 3 VO 4 ).…”

Section: Ctd Substrate Peptidesmentioning

confidence: 99%

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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Phosphorylation of RnA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YsPTsPs regulates eukaryotic transcription. Whereas ser5 is phosphorylated in the initiation phase, ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a ser5 CTD kinase that is unable to create ser2/ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate prephosphorylated at ser7 compared with the consensus hepta-repeat or the YsPTsPK variant. mass spectrometry reveals an equal number of phosphorylations to the number of heptarepeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.

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“…Worm extracts were sonicated and centrifuged to obtain soluble proteins. PKA activity was measured according to a previously reported protocol (90). Briefly, 30 g of protein was incubated with kinase assay buffer (50 mM Tris-HCl, 0.1 mM EGTA, and 10 mM magnesium acetate), Kemptide (PKA substrate peptide), and 1 mM [␥- 32 P]ATP at 30°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes

Lee

Han

Kong

et al. 2016

Journal of Biological Chemistry

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Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RI␣ and RII␤ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent serine/threonine kinase that mediates various cellular responses, including lipolysis. Since its discovery in the 1950s (1-4), the cAMP-PKA system has become one of the best understood signaling pathways in terms of biochemical properties. PKA is composed of catalytic subunits and cAMP-binding regulatory subunits (5). In the absence of cAMP stimulation, PKA forms an inactive tetramer composed of four subunits with two regulatory and two catalytic subunits. Upon nutritional deprivation or hormonal stimulation, activated adenylyl cyclase produces cAMP from ATP. Then, the regulatory subunit of PKA binds cAMP, causing a conformational change that decreases its affinity for catalytic subunits by ϳ10 4 -fold and leads to the release of active catalytic subunits to phosphorylate target proteins (6, 7).PKA has a wide range of substrates that regulate a number of physiological processes. To date, several hundred PKA target proteins have been identified, and yet new PKA substrates are continually being reported (8, 9). In mammalian adipocytes, numerous studies over the last 40 years have revealed that the cAMP-PKA axis forms a critical node in the regulation of lipolysis. For instance, major components of lipolytic pathways, including hormone-sensitive lipase (Hsl) 2 and perilipin 1 (Plin1), are direct targets of PKA (10, 11). Recently, adipose triglyceride lipase (Atgl) was also reported to be a target of PKA (12, 13). Upon receiving activation signals from molecules such as catecholamine and glucagon, activated PKA phosphorylates Plin1 and HSL to promote the recruitment of HSL to lipid droplets (14, 15). In addition, phosphorylated Plin1 releases comparative gene identification-58 (CGI-58; Abhd5), a coactivator of Atgl, to mediate lipolysis upon PKA activation (16 -20). On the contrary, insulin can activate phosphodiesterase 3B (Pde3b) and reduce cAMP levels (1...

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Assay of protein kinases using radiolabeled ATP: a protocol

Cited by 222 publications

References 11 publications

PAWS 1 controls Wnt signalling through association with casein kinase 1α

PAWS 1 controls Wnt signalling through association with casein kinase 1α

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes

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