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{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation.

Defilippi P, Rosso A, Dentelli P, Calvi C, Garbarino G, Tarone G, Pegoraro L, Brizzi MF - J. Cell Biol. (2005)

Bottom Line: Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry.Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression.Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126, Torino, Italy. paola.defilippi@unito.it

ABSTRACT
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

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The juxtamembrane domain of the IL-3R β common is required for STAT5A recruitment by cell adhesion. HEK293 cells expressing the Neo vector, the full-length IL-3R β common subunit (FL), and the myc-tagged Δ455 or Δ544 mutants of the IL-3R β common subunit were replated on FN-coated dishes for 20 min. (A) Cell extracts from Neo vector–transfected cells were IB with antibodies to phosphorylated STAT5A. (B and C) Cell extracts were IP with IL-3R β common antibodies (B) or mAb 9E10 to the Myc epitope tag (C), IB with phospho-STAT5 antibody or STAT5A antibody (top), and reimmunoblotted with IL-3R β common antibodies (B) or mAb 9E10 (C). (D) Nuclear cell extracts from HEK293 FL or Δ455 mutant, kept in suspension (S) or plated on FN, were subjected to EMSA analysis (left) and supershift assay (right). Arrows indicate the SIE complex and supershift species. (E) Extracts from FL or Δ455 mutant were IP with β1 integrin mAb and IB with mAb 9E10 (left) or IL-3R β common antibodies (right). As positive control (+), IL-3–treated M07-E cells were used. The results are representative of three independent experiments.
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fig5: The juxtamembrane domain of the IL-3R β common is required for STAT5A recruitment by cell adhesion. HEK293 cells expressing the Neo vector, the full-length IL-3R β common subunit (FL), and the myc-tagged Δ455 or Δ544 mutants of the IL-3R β common subunit were replated on FN-coated dishes for 20 min. (A) Cell extracts from Neo vector–transfected cells were IB with antibodies to phosphorylated STAT5A. (B and C) Cell extracts were IP with IL-3R β common antibodies (B) or mAb 9E10 to the Myc epitope tag (C), IB with phospho-STAT5 antibody or STAT5A antibody (top), and reimmunoblotted with IL-3R β common antibodies (B) or mAb 9E10 (C). (D) Nuclear cell extracts from HEK293 FL or Δ455 mutant, kept in suspension (S) or plated on FN, were subjected to EMSA analysis (left) and supershift assay (right). Arrows indicate the SIE complex and supershift species. (E) Extracts from FL or Δ455 mutant were IP with β1 integrin mAb and IB with mAb 9E10 (left) or IL-3R β common antibodies (right). As positive control (+), IL-3–treated M07-E cells were used. The results are representative of three independent experiments.

Mentions: To evaluate whether or not the intracellular region of the IL-3R β common may act as a STAT5A docking site, HEK293 cells were stably transfected with the full-length IL-3R β common subunit or one of two Myc-tagged deletion mutants, the first lacking the entire intracellular domain including the juxtamembrane region corresponding to the docking sites for JAK and STAT proteins (Δ455) and the second the intracellular region corresponding to the amino acids 545–881 (Δ544), still able to activate receptor signaling (Sakamaki et al., 1992). The basal adhesion-dependent activation of STAT5A was first evaluated in Neo vector-transfected cells. As shown in Fig. 5 A, the anti–phospho-STAT5 IB of total cell extracts revealed a faint band corresponding to the activated STAT5, indicating that a minimal STAT5A activation is detectable in these cells upon adhesion. IL-3–treated M07-E cells were used as positive control (Fig. 5 A, +). Subsequently, coimmunoprecipitation experiments were performed on HEK293 cells expressing the full-length IL-3R β common or one of the two mutants. As shown in Fig. 5 B, in agreement with the data obtained in primary endothelial cells, in response to adhesion tyrosine-phosphorylated STAT5A coimmunoprecipitated with the IL-3R β common in HEK293 cells expressing the full-length receptor. In contrast, phosphorylated STAT5A was undetectable in the immunoprecipitates of the Δ455 truncated form of the IL-3R β common (Fig. 5 C). Also in these experiments IL-3–treated M07-E cells were used as positive control. Consistent with the ability of Δ544 mutant receptor to trigger intracellular signaling (Sakamaki et al., 1992), it was still able to recruit STAT5A (Fig. 5 C).


{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation.

Defilippi P, Rosso A, Dentelli P, Calvi C, Garbarino G, Tarone G, Pegoraro L, Brizzi MF - J. Cell Biol. (2005)

The juxtamembrane domain of the IL-3R β common is required for STAT5A recruitment by cell adhesion. HEK293 cells expressing the Neo vector, the full-length IL-3R β common subunit (FL), and the myc-tagged Δ455 or Δ544 mutants of the IL-3R β common subunit were replated on FN-coated dishes for 20 min. (A) Cell extracts from Neo vector–transfected cells were IB with antibodies to phosphorylated STAT5A. (B and C) Cell extracts were IP with IL-3R β common antibodies (B) or mAb 9E10 to the Myc epitope tag (C), IB with phospho-STAT5 antibody or STAT5A antibody (top), and reimmunoblotted with IL-3R β common antibodies (B) or mAb 9E10 (C). (D) Nuclear cell extracts from HEK293 FL or Δ455 mutant, kept in suspension (S) or plated on FN, were subjected to EMSA analysis (left) and supershift assay (right). Arrows indicate the SIE complex and supershift species. (E) Extracts from FL or Δ455 mutant were IP with β1 integrin mAb and IB with mAb 9E10 (left) or IL-3R β common antibodies (right). As positive control (+), IL-3–treated M07-E cells were used. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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fig5: The juxtamembrane domain of the IL-3R β common is required for STAT5A recruitment by cell adhesion. HEK293 cells expressing the Neo vector, the full-length IL-3R β common subunit (FL), and the myc-tagged Δ455 or Δ544 mutants of the IL-3R β common subunit were replated on FN-coated dishes for 20 min. (A) Cell extracts from Neo vector–transfected cells were IB with antibodies to phosphorylated STAT5A. (B and C) Cell extracts were IP with IL-3R β common antibodies (B) or mAb 9E10 to the Myc epitope tag (C), IB with phospho-STAT5 antibody or STAT5A antibody (top), and reimmunoblotted with IL-3R β common antibodies (B) or mAb 9E10 (C). (D) Nuclear cell extracts from HEK293 FL or Δ455 mutant, kept in suspension (S) or plated on FN, were subjected to EMSA analysis (left) and supershift assay (right). Arrows indicate the SIE complex and supershift species. (E) Extracts from FL or Δ455 mutant were IP with β1 integrin mAb and IB with mAb 9E10 (left) or IL-3R β common antibodies (right). As positive control (+), IL-3–treated M07-E cells were used. The results are representative of three independent experiments.
Mentions: To evaluate whether or not the intracellular region of the IL-3R β common may act as a STAT5A docking site, HEK293 cells were stably transfected with the full-length IL-3R β common subunit or one of two Myc-tagged deletion mutants, the first lacking the entire intracellular domain including the juxtamembrane region corresponding to the docking sites for JAK and STAT proteins (Δ455) and the second the intracellular region corresponding to the amino acids 545–881 (Δ544), still able to activate receptor signaling (Sakamaki et al., 1992). The basal adhesion-dependent activation of STAT5A was first evaluated in Neo vector-transfected cells. As shown in Fig. 5 A, the anti–phospho-STAT5 IB of total cell extracts revealed a faint band corresponding to the activated STAT5, indicating that a minimal STAT5A activation is detectable in these cells upon adhesion. IL-3–treated M07-E cells were used as positive control (Fig. 5 A, +). Subsequently, coimmunoprecipitation experiments were performed on HEK293 cells expressing the full-length IL-3R β common or one of the two mutants. As shown in Fig. 5 B, in agreement with the data obtained in primary endothelial cells, in response to adhesion tyrosine-phosphorylated STAT5A coimmunoprecipitated with the IL-3R β common in HEK293 cells expressing the full-length receptor. In contrast, phosphorylated STAT5A was undetectable in the immunoprecipitates of the Δ455 truncated form of the IL-3R β common (Fig. 5 C). Also in these experiments IL-3–treated M07-E cells were used as positive control. Consistent with the ability of Δ544 mutant receptor to trigger intracellular signaling (Sakamaki et al., 1992), it was still able to recruit STAT5A (Fig. 5 C).

Bottom Line: Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry.Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression.Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126, Torino, Italy. paola.defilippi@unito.it

ABSTRACT
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

Show MeSH
Related in: MedlinePlus