Detection of phosphorylated T and B cell antigen receptor species by Phos-tag SDS- and Blue Native-PAGE

Deswal, Sumit; Beck-García, Katharina; Blumenthal, Britta; Dopfer, Elaine P.; Schamel, Wolfgang W. A.

doi:10.1016/j.imlet.2009.12.012

Cited by 14 publications

(10 citation statements)

References 39 publications

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“…Thus, phosphorylated proteins appear as double or multiple bands following immunoblotting, depending on the phosphorylation degree. Phos-Tag technology has been used successfully in numerous cases for animal proteins (e.g., Kinoshita et al, 2006Kinoshita et al, , 2009Gallon, 2008;Oh and Irvine, 2008;Sikkema et al, 2009;Deswal et al, 2009) and also in plant protein phosphorylation studies (e.g., Bethke et al, 2009;Panteris et al, 2010). This assay was performed in extracts from mutant or wild-type Arabidopsis plants obtained from the homogenization of liquid nitrogen powders in G-buffer from which b-glycerophosphate was omitted because it would interfere with PhosTag mobility retardation (Kinoshita et al, 2006).…”

Section: Analysis Of Phosphorylated Proteinsmentioning

confidence: 99%

ArabidopsisHomologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

et al. 2010

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A double homozygous recessive mutant in the Arabidopsis thaliana homologs of nucleus-and phragmoplast-localized kinase 2 (ANP2) and 3 (ANP3) genes and a homozygous recessive mutant in the mitogen-activated protein kinase 4 (MPK4) gene of Arabidopsis exhibit deficiencies in the overall microtubule (MT) organization, which result in abnormal cell growth patterns, such as branching of root hairs and swelling of diffusely growing epidermal cells. Genetic, pharmacological, molecular, cytological, and biochemical analyses show that the major underlying mechanism for these phenotypes is excessive MT stabilization manifested in both mutants as heavy MT bundling, disorientation, and drug stability. The above defects in MAPK signaling result in the adverse regulation of members of the microtubule-associated protein (MAP65) protein family, including strongly diminished phosphorylation of MAP65-1. These data suggest that ANP2/ANP3, MPK4, and the microtubule-associated protein MAP65-1, a putative target of MPK4 signaling, are all essential for the proper organization of cortical microtubules in Arabidopsis epidermal cells.

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Section: Analysis Of Phosphorylated Proteinsmentioning

confidence: 99%

ArabidopsisHomologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

et al. 2010

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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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“…IP was done using 2 µg of the respective antibody per sample and 10 µl of protein G-coupled sepharose beads (GE Healthcare Bio-Sciences AB) overnight by rotating at 4°C [45]. After IP, immunoprecipitates were washed three times with lysis buffer and boiled in reducing sample buffer at 95°C for 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

et al. 2011

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BackgroundTo understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success.Methodology/Principal FindingsWe use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally.Conclusions/SignificanceThe quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network.

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Detection of phosphorylated T and B cell antigen receptor species by Phos-tag SDS- and Blue Native-PAGE

Cited by 14 publications

References 39 publications

ArabidopsisHomologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

ArabidopsisHomologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

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

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks

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