A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation

Min Ying Zhang, Dwayne L. Barber, Dario R. Alessi, Laurie L. Bell, Carol Stine, Melody H.H. Nguyen, Bryan K. Beattie, Joseph Y. Cheung, Barbara Miller

Research output: Contribution to journalArticle

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Abstract

Objective. Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined. Materials and Methods. Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined.ResultsPhosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba/F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. Conclusions. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.

Original languageEnglish (US)
Pages (from-to)432-440
Number of pages9
JournalExperimental Hematology
Volume29
Issue number4
DOIs
StatePublished - Apr 25 2001

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Erythropoietin Receptors
70-kDa Ribosomal Protein S6 Kinases
Erythropoietin
Phosphorylation
Phosphatidylinositols
Protein Kinases
Intercellular Signaling Peptides and Proteins
3-Phosphoinositide-Dependent Protein Kinases

All Science Journal Classification (ASJC) codes

  • Hematology
  • Molecular Biology
  • Genetics
  • Cell Biology
  • Cancer Research

Cite this

Zhang, Min Ying ; Barber, Dwayne L. ; Alessi, Dario R. ; Bell, Laurie L. ; Stine, Carol ; Nguyen, Melody H.H. ; Beattie, Bryan K. ; Cheung, Joseph Y. ; Miller, Barbara. / A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation. In: Experimental Hematology. 2001 ; Vol. 29, No. 4. pp. 432-440.
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title = "A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation",
abstract = "Objective. Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined. Materials and Methods. Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined.ResultsPhosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba/F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. Conclusions. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.",
author = "Zhang, {Min Ying} and Barber, {Dwayne L.} and Alessi, {Dario R.} and Bell, {Laurie L.} and Carol Stine and Nguyen, {Melody H.H.} and Beattie, {Bryan K.} and Cheung, {Joseph Y.} and Barbara Miller",
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doi = "10.1016/S0301-472X(00)00681-0",
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volume = "29",
pages = "432--440",
journal = "Experimental Hematology",
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Zhang, MY, Barber, DL, Alessi, DR, Bell, LL, Stine, C, Nguyen, MHH, Beattie, BK, Cheung, JY & Miller, B 2001, 'A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation', Experimental Hematology, vol. 29, no. 4, pp. 432-440. https://doi.org/10.1016/S0301-472X(00)00681-0

A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation. / Zhang, Min Ying; Barber, Dwayne L.; Alessi, Dario R.; Bell, Laurie L.; Stine, Carol; Nguyen, Melody H.H.; Beattie, Bryan K.; Cheung, Joseph Y.; Miller, Barbara.

In: Experimental Hematology, Vol. 29, No. 4, 25.04.2001, p. 432-440.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation

AU - Zhang, Min Ying

AU - Barber, Dwayne L.

AU - Alessi, Dario R.

AU - Bell, Laurie L.

AU - Stine, Carol

AU - Nguyen, Melody H.H.

AU - Beattie, Bryan K.

AU - Cheung, Joseph Y.

AU - Miller, Barbara

PY - 2001/4/25

Y1 - 2001/4/25

N2 - Objective. Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined. Materials and Methods. Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined.ResultsPhosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba/F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. Conclusions. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.

AB - Objective. Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined. Materials and Methods. Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined.ResultsPhosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba/F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. Conclusions. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.

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U2 - 10.1016/S0301-472X(00)00681-0

DO - 10.1016/S0301-472X(00)00681-0

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