Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins

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

doi:10.1074/mcp.t500024-mcp200

Cited by 1,017 publications

(963 citation statements)

References 31 publications

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“…Phos-tag gels separate phosphorylated from unphosphorylated forms of proteins [16], a property we validated by using Antarctic phosphatase-treated recombinant RprY as a positive control (Figure 6(a), lane 7); this treatment also confirmed that RprY purified from E. coli was already phosphorylated (Figure 6(a), lanes 1 and 7). Phosphorylated RprY was incubated with Ac-CoA to examine whether acetylation affected phosphorylation, and as shown in Figure 6(a) (lanes 2–4), the increased levels of non-phosphorylated RprY in Phos-tag gels in the presence of increasing amounts of Ac-CoA indicated that acetylation of RprY resulted in dephosphorylation, and possibly inactivation, of the regulator.…”

Section: Resultsmentioning

confidence: 76%

Post-translational regulation of a Porphyromonas gingivalis regulator

Krishnan

Duncan³

2018

Journal of Oral Microbiology

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Background: Bacteria use two-component signal transduction systems (among others) to perceive and respond to environmental changes. Within the genus Porphyromonas, we observed degeneration of these systems, as exemplified by the loss of RprX, the sensor kinase partner of the RprY.Objective: The purpose of this study was to investigate modulation of RprY function by acetylation.Design: The transcriptional activity of the rprY-pat genes were measured by RT-PCR and 5ʹ-RACE. The acetylation of RprY were detected by western blotting. Electromobility shift and in vitro ChIP assays were used to measure the DNA binding activity of RprY. The expression of RprY target genes was measured by qRT-PCR. Effects of acetylation on phosphorylation of RprY were measured by Phos-tag gels.Results: The rprY gene is cotranscribed with pat. RprY is acetylated in vivo, and autoacetylated in vitro in a reaction that is enhanced by Pat; the CobB sirtuin deacetylates RprY. Acetylation reduced the DNA binding of RprY. Induced oxidative stress decreased production of RprY in vivo, increased its acetylation and increased expression of nqrA.Conclusions: We propose that to compensate for the loss of RprX, P. gingivalis has evolved a novel mechanism to inactivate RprY through acetylation.

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Section: Resultsmentioning

confidence: 76%

Post-translational regulation of a Porphyromonas gingivalis regulator

Krishnan

Duncan³

2018

Journal of Oral Microbiology

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“…To examine whether CTCF is also phosphorylated in the mitotic phase, we carried out phos-tag SDS/PAGE [16] which relies on the fact that phosphorylated proteins migrate slower than unphosphorylated proteins in the phos-tag gel. CTCF in mitotic cell lysates migrated slower than that in asynchronous cell lysates on phos-tag SDS/PAGE (Fig.…”

Section: Phosphorylation Of Ctcf In Mitosismentioning

confidence: 99%

Mitotic phosphorylation of CCCTC‐binding factor (CTCF) reduces its DNA binding activity

et al. 2017

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During mitosis, higher order chromatin structures are disrupted and chromosomes are condensed to achieve accurate chromosome segregation. CCCTC‐binding factor (CTCF) is a highly conserved and ubiquitously expressed C2H2‐type zinc finger protein which is considered to be involved in epigenetic memory through regulation of higher order chromatin architecture. However, the regulatory mechanism of CTCF in mitosis is still unclear. Here we found that the DNA‐binding activity of CTCF is regulated in a phosphorylation‐dependent manner during mitosis. The linker domains of the CTCF zinc finger domain were found to be phosphorylated during mitosis. The phosphorylation of linker domains impaired the DNA‐binding activity in vitro. Mutation analyses showed that amino acid residues (Thr289, Thr317, Thr346, Thr374, Ser402, Ser461, and Thr518) located in the linker domains were phosphorylated during mitosis. Based on these results, we propose that the mitotic phosphorylation of the linker domains of CTCF is important for the dissociation of CTCF from mitotic chromatin.

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“…To test this hypothesis, we first examined whether CK1 phosphorylates Ci A in vivo by monitoring the mobility shift of HA-Ci -PKA in S2 cells in the absence or presence of CK1* using the phospho-tag gel that specifically retards phosphorylated proteins (48). We found that coexpression of CK1* induced a mobility shift of HA-Ci -PKA but not HA-Cim1-6 (Fig.…”

Section: Ck1 Inhibits Hib Binding To CI By Phosphorylating Multiple Smentioning

confidence: 99%

Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator

Shi¹,

Li²,

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Hedgehog (Hh) signaling governs many developmental processes by regulating the balance between the repressor (Ci R /Gli R ) and activator (Ci A /Gli A ) forms of Cubitus interruptus (Ci)/gliomaassociated oncogene homolog (Gli) transcription factors. Although much is known about how Ci R /Gli R is controlled, the regulation of Ci A /Gli A remains poorly understood. Here we demonstrate that Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 and Suppressor of fused (Sufu) by protecting Ci A from premature degradation. We show that Hh stimulates Ci phosphorylation by CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by the Hh-induced MATH and BTB domain containing protein (HIB), a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases. In Hh-receiving cells, reduction of CK1 activity accelerated HIB-mediated degradation of Ci A , leading to premature loss of pathway activity. We also provide evidence that Gli A is regulated by CK1 in a similar fashion and that CK1 acts downstream of Sufu to promote Sonic hedgehog signaling. Taken together, our study not only reveals an unanticipated and conserved mechanism by which phosphorylation of Ci/Gli positively regulates Hh signaling but also provides the first evidence, to our knowledge, that substrate recognition by the Cullin 3 family of E3 ubiquitin ligases is negatively regulated by a kinase.

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Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins

Cited by 1,017 publications

References 31 publications

Post-translational regulation of a Porphyromonas gingivalis regulator

Post-translational regulation of a Porphyromonas gingivalis regulator

Mitotic phosphorylation of CCCTC‐binding factor (CTCF) reduces its DNA binding activity

Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator

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