Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE

Kinoshita, Eiji; Kinoshita–Kikuta, Emiko; Koike, Takao

doi:10.1038/nprot.2009.154

Cited by 347 publications

(309 citation statements)

References 49 publications

Supporting

Mentioning

303

Contrasting

Order By: Relevance

“…The main reason is that, since Phos-tag retards the phosphoprotein during electrophoresis, it also hampers its subsequent transfer to a membrane, leading to incomplete transfer of the phosphorylated form and, therefore, to an underestimation of phosphorylation. This problem can be partly alleviated by incubating the gel in EDTA-containing solution to strip Zn 2+ prior to the transfer (Kinoshita et al, 2009). Here, we use an improved protocol with a two-layer resolving gel containing the Phos-tag ligand only in the upper layer.…”

Section: An Improved Phos-tag Page Protocol Allows a Quantitative Meamentioning

confidence: 99%

Phosphorylation of the Lhcb2 isoform of Light Harvesting Complex II is central to state transitions

et al. 2015

View full text Add to dashboard Cite

ORCID IDs: 0000-0003-0587-7621 (P.L.); 0000-0002-6155-9153 (D.D.).Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (.98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.

show abstract

Section: An Improved Phos-tag Page Protocol Allows a Quantitative Meamentioning

confidence: 99%

Phosphorylation of the Lhcb2 isoform of Light Harvesting Complex II is central to state transitions

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Gels were stained with Coomassie Brilliant Blue and analyzed by autoradiography. The Phos-tag gels were made as described (Kinoshita et al, 2009) …”

Section: In Vitro Phosphorylation Assaymentioning

confidence: 99%

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

et al. 2017

View full text Add to dashboard Cite

Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo.

show abstract

“…As described previously (39), cell lysates were separated by SDS-PAGE using 8% acrylamide gels including 15 μM Phos-tag acrylamide (Nard Chemicals, Hiroshima, Japan) and 30 μM MnCl 2 . After electrophoresis, the gels were washed in transfer buffer containing 1 mM EDTA for 10 min and then transferred onto PVDF membranes as described (26). The membranes were treated, and the bands were visualized as described above.…”

Section: Phosphorylation Of Gcma On Serines 328 378 and 383 Contribumentioning

confidence: 99%

PMA-induced GCMa phosphorylation stimulates its transcriptional activity and degradation

et al. 2012

View full text Add to dashboard Cite

Glial cells missing Drosophila homolog a (GCMa) is a member of the GCM transcription factor family and plays critical roles in trophoblast differentiation and placental functions. It is well established that the cyclic AMP (cAMP)-dependent pathway induces the expression and transcriptional activity of GCMa by regulating post-translational modifications of GCMa, which results in enhancement of trophoblast differentiation. We previously observed that phorbol 12-myristate 13-acetate (PMA) stimulates phosphorylation of GCMa on serines 328, 378 and 383 through the protein kinase C (PKC)-and mitogen-activated protein kinase kinase (MEK)/extracellular signalregulated kinase (ERK)-dependent pathway, which decreases the protein stability of GCMa. Here we report that PMA increases the ubiquitination level of GCMa, dependent on the phosphorylation of GCMa on serines 328, 378 and 383. We found that this phosphorylation also stimulates the transcriptional activity of GCMa. Our data indicate that the PMA-induced PKC-and MEK/ERKdependent pathway enhances the degradation as well as the transcriptional activity of GCMa. We also examined the impact of this signaling pathway on trophoblasts and the results suggest that the PKC-and MEK/ERK-dependent pathway is involved in the regulation of trophoblast differentiation.

show abstract

Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE

Cited by 347 publications

References 49 publications

Phosphorylation of the Lhcb2 isoform of Light Harvesting Complex II is central to state transitions

Phosphorylation of the Lhcb2 isoform of Light Harvesting Complex II is central to state transitions

Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response

PMA-induced GCMa phosphorylation stimulates its transcriptional activity and degradation

Contact Info

Product

Resources

About