A Cellular Reporter Assay to Monitor Insulin Receptor Kinase Activity Based on STAT 5-Dependent Luciferase Gene Expression

Störz, Peter; Döppler, Heike; Horn-Müller, Judith; Groner, Bernd; Pfizenmaier, Klaus; Ga, Müller

doi:10.1006/abio.1999.4345

Cited by 22 publications

(11 citation statements)

References 42 publications

(37 reference statements)

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“…The results of our study conflict with reports that suggest that INS is a potent inducer of STAT5B (4, 5, 6, 20). Our results also do not support recent studies that suggest that SOCS‐3 inhibits INS action (7, 11).…”

Section: Discussioncontrasting

confidence: 99%

“…STAT5 proteins have been shown to be activated by growth hormone (GH) in many cell types. However, some recent studies also suggest that STAT5B is a physiological substrate of the INS receptor in a variety of cells types (4, 5, 6). Two of these studies suggest that INS activation of STAT5B is independent of JAK2 kinase, which is normally involved in STAT5 phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

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Modulation and Lack of Cross‐Talk between Signal Transducer and Activator of Transcription 5 and Suppressor of Cytokine Signaling‐3 in Insulin and Growth Hormone Signaling in 3T3‐L1 Adipocytes

Story

Stephens

2006

Obesity

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Research Methods and Procedures:Fully differentiated 3T3-L1 adipocytes were exposed to INS, growth hormone (GH), or both of these growth factors, and the activation of STAT5 proteins and mitogen-activated protein kinase was examined using phospho-specific antibodies. The induction of SOCS-3 mRNA was assessed by Northern blot analysis. INS-stimulated glucose transport was also measured. Results: We observed that GH, not INS, induced STAT5 activation in adipocytes in a manner that was independent of extracellular signal-regulated kinase (ERK) activation or new protein synthesis. GH strongly induced SOCS-3 mRNA expression, whereas INS had a much less potent effect on SOCS-3 mRNA expression. Because SOCS-3 has been implicated in the attenuation of GH and INS signaling, we examined the cross-talk between these signaling pathways. GH pretreatment of adipocytes inhibited GH signaling. Similarly, INS pretreatment inhibited INS signaling. However, INS did not block the GH-induced activation of STAT5, and GH did not block the INS induction of ERK activity or of increased glucose uptake. We observed that neither new protein synthesis nor activation of ERKs 1 and 2 were required for the inhibition of GH signaling. Discussion: These results demonstrate that blocking the induction of the SOCS-3 protein has no effect on the attenuation of GH signaling and support recent studies suggesting that SOCS proteins have additional functions. In addition, these studies demonstrate that GH-induced SOCS-3 expression is insufficient to inhibit INS-induced glucose uptake in adipocytes.Key words: signal transducer and activator of transcription-5, suppressor of cytokine signaling-3, adipocytes, extracellular signal-regulated kinases 1 and 2, mitogenactivated protein kinase

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Section: Discussioncontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modulation and Lack of Cross‐Talk between Signal Transducer and Activator of Transcription 5 and Suppressor of Cytokine Signaling‐3 in Insulin and Growth Hormone Signaling in 3T3‐L1 Adipocytes

Story

Stephens

2006

Obesity

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“…STAT5 or STAT3 activation was assessed by measuring luciferase production of cells transfected with the pLHRE-luc 23,24 or pGL3bPpr2-luc constructs. 25 Mitogen-activated protein kinase (MAPK) activation was assessed with the pSRE-luc construct.…”

Section: Dual Luciferase Assaysmentioning

confidence: 99%

An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor

Staerk

Lacout

Sato

et al. 2006

Blood

144

169

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Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (⌬5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, ⌬5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. ⌬5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the ⌬5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces. IntroductionThe thrombopoietin receptor (c-mpl or TpoR) and its ligand thrombopoietin (Tpo) regulate the proliferation of megakaryocytic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. 1,2 Tpo or TpoR knockout mice exhibit a significant reduction of megakaryocytes and circulating platelets, and they also show reduced number of hematopoietic stem cells (HSCs) in the bone marrow, indicating a role for the TpoR and its ligand in HSC self-renewal. 3 Like the erythropoietin receptor (EpoR), the TpoR is thought to function as a homodimer after binding Tpo. Downstream signaling pathways activated by the TpoR are triggered by 2 cytoplasmic tyrosine kinases, the Janus kinase (Jak) 2, and Tyk2. [4][5][6] Upon receptor activation, phosphorylated tyrosine residues in the cytoplasmic domain of the TpoR and in Jaks provide docking sites for the SH2 domains of many signaling proteins, such as STAT3 and STAT5 (the signal transducers and activators of transcription 3 and 5), shc, SHIP, Grb2, and PI3K. 4,5,[7][8][9] Several activating mutations in the TpoR have been identified. In one, the envelope protein of the myeloproliferative virus replaces the extracellular domain of the receptor and activates it by oligomerization. 10 Introduction of a cysteine residue in the extracellular domain of the TpoR at a position equivalent to that of the constitutively active R129C EpoR mutant 11,12 leads to active disulfide-bonded TpoR dimers. 13 Replacement of S498 (or S505) in the transmembrane (TM) domain by asparagine results in constitutively active receptors, 9,14 most likely by ligand-independent dimerization. 9,10,15 Deletion of the membrane distal extracellular cytokine receptor module of the TpoR results...

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“…Several different platforms such as reporter assays 5,6 and fluorescence resonance energy transfer assays 7,8 have been developing for screening small molecular compounds that display kinase inhibition. These assays often utilize recombinant cells to detect the catalytic activity of the target kinase by converting the catalytic activity to a specific readout.…”

Section: Introductionmentioning

confidence: 99%

Dual Enzyme-Linked Immunosorbent Assay System for Detection of Endogenous Kinase Activities of Mitogen- and Stress-Activated Protein Kinase-1/2

Ishii

Sootome²,

Toyoda³

et al. 2007

ASSAY and Drug Development Technologies

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The kinase signaling cascades related to mitogen- and stress-activated protein kinase-1 and -2 (MSK1 and MSK2, respectively) are attractive targets for pharmaceutical intervention, especially for neural injury. Therefore, we have developed a high throughput and cost-effective detection platform for measuring selective activity of MSK1/MSK2 in cells. Through the serial monitoring of both the p38 mitogen-activated protein kinase (stress-activated protein kinase 2B)-MSK1/MSK2- cyclic AMP response element binding protein (CREB)/activating transcription factor 1 (ATF1) pathway and the p38-mammalian heat shock protein 27 (Hsp27) pathway in HeLa cells treated with anisomycin, two selective MSK1 inhibitors showed inhibition of CREB (Ser-133) and ATF1 (Ser-63) phosphorylation and no interference with Hsp-27 phosphorylation (Ser-82). On the other hand, the p38 inhibitor SB-220025 showed equipotent inhibition of CREB/ATF1 and Hsp27 phosphorylation. This study demonstrated that the specific inhibition of a target kinase could be subsequently monitored by a secondary assay that measures the intervention arising from the modulation of off-target kinases. Our established system is applicable to inhibitor screening and drug discovery related to MSK1/MSK2.

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A Cellular Reporter Assay to Monitor Insulin Receptor Kinase Activity Based on STAT 5-Dependent Luciferase Gene Expression

Cited by 22 publications

References 42 publications

Modulation and Lack of Cross‐Talk between Signal Transducer and Activator of Transcription 5 and Suppressor of Cytokine Signaling‐3 in Insulin and Growth Hormone Signaling in 3T3‐L1 Adipocytes

Modulation and Lack of Cross‐Talk between Signal Transducer and Activator of Transcription 5 and Suppressor of Cytokine Signaling‐3 in Insulin and Growth Hormone Signaling in 3T3‐L1 Adipocytes

An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor

Dual Enzyme-Linked Immunosorbent Assay System for Detection of Endogenous Kinase Activities of Mitogen- and Stress-Activated Protein Kinase-1/2

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