Detection of ERK1/2 Activities Using Affinity Reagents

Pulido, Rafael; Zúñiga, Ángel; Ullrich, Axel

doi:10.1385/1-59259-671-1:49

Cited by 3 publications

(4 citation statements)

References 3 publications

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“…Since PT‐ERK was detected mainly in the cytoplasm, the involved PTP should also be localized in this region. This conclusion is in agreement with the recent reports that PTP‐SL, STEP [26] and PTP‐ER [27] are localized in cytoplasm. Similarly, the existence of PY‐ERKs in the nucleus indicates that the involved protein Ser/Thr phosphatases should be localized, at least in part, in the nucleus.…”

Section: Resultssupporting

confidence: 94%

“…3, 193) is likely to occur by constitutively active protein Ser/Thr phosphatases, which can remove the phosphate from the Thr but not the Tyr residues of ERKs, and which have been reported to play a role in the inactivation process of ERK [15]. In addition, the existence of a significant amount of mono‐phosphorylated PT‐ERK described above and the sharp decline in the amount of PY‐ERK in the absence of significant MEK activity indicate that as suggested [26], PTP is also involved in the mechanism of ERK dephosphorylation. The accumulation of PY‐ERK2 6 min after stimulation, and the slower time course of appearance of mono‐phosphorylated PT, would predict that the cellular activity of this putative PTP is lower than that of the protein Ser/Thr phosphatase.…”

Section: Resultsmentioning

confidence: 78%

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Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

Yao

Dolginov²,

Hanoch

et al. 2000

FEBS Letters

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When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.z 2000 Federation of European Biochemical Societies.

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Section: Resultssupporting

confidence: 94%

Section: Resultsmentioning

confidence: 78%

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

Yao

Dolginov²,

Hanoch

et al. 2000

FEBS Letters

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“…So far, MAPK dephosphorylation has been attributed to three classes of phosphatases: (i) serine/threonine‐specific phosphatases [17], (ii) tyrosine‐specific phosphatases [18,19], and (iii) dual‐specificity phosphatases that dephosphorylate both threonine and tyrosine residues [20]. The result described above showed that the light‐activated phosphatase dephosphorylates either or both of the regulatory tyrosine and threonine residues.…”

Section: Resultsmentioning

confidence: 93%

Circadian and photic regulation of MAP kinase by Ras‐ and protein phosphatase‐dependent pathways in the chick pineal gland

2001

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Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation. ß

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“…Recently, some ERK substrates have been shown to contain motifs that mediate high affinity interactions with ERK. Such examples are a docking site D‐domain in phosphatases [36,37], transcription factors [38], phosphodiesterase [39] and kinases [40] and an FXFP motif in ETS transcription factors of the Elk subfamily [38,40]. The region required for the direct ERK–RAGE interaction has a similarity to the D‐domain‐like ERK docking sites of MnK1/2, MSK1, and RSKs, which are characterized by a cluster of basic residues [39–43].…”

Section: Discussionmentioning

confidence: 99%

The receptor for advanced glycation end‐products (RAGE) directly binds to ERK by a D‐domain‐like docking site

Ishihara¹,

Tsutsumi²,

Kawane³

et al. 2003

FEBS Letters

178

135

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The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-U UB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of in£ammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were puri¢ed from rat lung extracts using an a⁄nity chromatography technique and identi¢ed to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were con¢rmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK^RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen-and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE. ß

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Detection of ERK1/2 Activities Using Affinity Reagents

Cited by 3 publications

References 3 publications

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

Circadian and photic regulation of MAP kinase by Ras‐ and protein phosphatase‐dependent pathways in the chick pineal gland

The receptor for advanced glycation end‐products (RAGE) directly binds to ERK by a D‐domain‐like docking site

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