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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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FN-dependent STAT5A phosphorylation and recruitment to the IL-3R β common depends on integrin-mediated JAK2 kinase activity. (A) Extracts from M07-E cells, untreated or treated with IL-3 with (+) or without (−) 100 nM of JAK2 kinase inhibitor AG490, were IP with JAK2 antibody, IB with anti-PY, and reimmunoblotted with JAK2 antibodies. (B and C) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) 100 nM AG490 were IP with IL-3R β common antibodies, divided in two aliquots, and run on 8% SDS-PAGE. (B) Filter was IB with phospho-STAT5 antibody or STAT5A antibody (top) and reimmunoblotted with IL-3R β common antibodies (bottom). (C) Immunoblotting was performed with anti-PY antibodies (top) or IL-3R β common antibodies. (D) Cell extracts from endothelial cells were IP with β1 integrin mAb TS2/16 and IB with JAK2 antibodies (top) or IL-3R β common antibody (bottom). (E) Cell extracts from endothelial cells with (+) or without (−) IL-3 were IP with JAK2 antibodies and subjected to SDS-PAGE. Filters were IB with anti-PY antibodies or JAK2 antibodies. The results are representative of three independent experiments.
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fig4: FN-dependent STAT5A phosphorylation and recruitment to the IL-3R β common depends on integrin-mediated JAK2 kinase activity. (A) Extracts from M07-E cells, untreated or treated with IL-3 with (+) or without (−) 100 nM of JAK2 kinase inhibitor AG490, were IP with JAK2 antibody, IB with anti-PY, and reimmunoblotted with JAK2 antibodies. (B and C) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) 100 nM AG490 were IP with IL-3R β common antibodies, divided in two aliquots, and run on 8% SDS-PAGE. (B) Filter was IB with phospho-STAT5 antibody or STAT5A antibody (top) and reimmunoblotted with IL-3R β common antibodies (bottom). (C) Immunoblotting was performed with anti-PY antibodies (top) or IL-3R β common antibodies. (D) Cell extracts from endothelial cells were IP with β1 integrin mAb TS2/16 and IB with JAK2 antibodies (top) or IL-3R β common antibody (bottom). (E) Cell extracts from endothelial cells with (+) or without (−) IL-3 were IP with JAK2 antibodies and subjected to SDS-PAGE. Filters were IB with anti-PY antibodies or JAK2 antibodies. The results are representative of three independent experiments.

Mentions: We previously demonstrated that integrin-mediated adhesion triggered JAK2 activation (Brizzi et al., 1999). Here, we investigated the role of JAK2 in the formation of the β1 integrin–IL-3R β common–STAT5A complex (Fig. 2 C). As positive control for the inhibitory effect of the JAK2 inhibitor AG-490, M07-E cells were used (Fig. 4 A). Subsequently, endothelial cells pretreated with 100 nM were kept in suspension or plated on FN and cell extracts were IP with the antiserum to the IL-3R β common. The anti–phospho-STAT5 immunoblot in Fig. 4 B shows that the phosphorylated STAT5A did not coimmunoprecipitate with the IL-3R β common in adherent cells pretreated with AG-490. In addition, the finding that STAT5A cannot be detected in the IL-3R β common immunoprecipitate from cells pretreated with AG-490 sustains the possibility that JAK2 kinase activity regulates both STAT5 recruitment and activation (Fig. 4 B). Similarly, in the immunoprecipitates of the IL-3R β common, the phosphorylated bands corresponding to STAT5A and the IL-3R β common were not present when the cells were pretreated with AG490 (Fig. 4 C). Moreover, by immunoprecipitating with antibodies to the β1 integrin, JAK2 was found associated with the β1 integrin–IL-3R β common complex only in cells adherent to FN (Fig. 4 D). Adhesion to FN also triggers JAK2 tyrosine phosphorylation (Fig. 4 E). These data support a potential role of JAK2 in regulating both adhesion-mediated IL-3R β common phosphorylation and STAT5A recruitment and activation.


{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)

FN-dependent STAT5A phosphorylation and recruitment to the IL-3R β common depends on integrin-mediated JAK2 kinase activity. (A) Extracts from M07-E cells, untreated or treated with IL-3 with (+) or without (−) 100 nM of JAK2 kinase inhibitor AG490, were IP with JAK2 antibody, IB with anti-PY, and reimmunoblotted with JAK2 antibodies. (B and C) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) 100 nM AG490 were IP with IL-3R β common antibodies, divided in two aliquots, and run on 8% SDS-PAGE. (B) Filter was IB with phospho-STAT5 antibody or STAT5A antibody (top) and reimmunoblotted with IL-3R β common antibodies (bottom). (C) Immunoblotting was performed with anti-PY antibodies (top) or IL-3R β common antibodies. (D) Cell extracts from endothelial cells were IP with β1 integrin mAb TS2/16 and IB with JAK2 antibodies (top) or IL-3R β common antibody (bottom). (E) Cell extracts from endothelial cells with (+) or without (−) IL-3 were IP with JAK2 antibodies and subjected to SDS-PAGE. Filters were IB with anti-PY antibodies or JAK2 antibodies. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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fig4: FN-dependent STAT5A phosphorylation and recruitment to the IL-3R β common depends on integrin-mediated JAK2 kinase activity. (A) Extracts from M07-E cells, untreated or treated with IL-3 with (+) or without (−) 100 nM of JAK2 kinase inhibitor AG490, were IP with JAK2 antibody, IB with anti-PY, and reimmunoblotted with JAK2 antibodies. (B and C) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) 100 nM AG490 were IP with IL-3R β common antibodies, divided in two aliquots, and run on 8% SDS-PAGE. (B) Filter was IB with phospho-STAT5 antibody or STAT5A antibody (top) and reimmunoblotted with IL-3R β common antibodies (bottom). (C) Immunoblotting was performed with anti-PY antibodies (top) or IL-3R β common antibodies. (D) Cell extracts from endothelial cells were IP with β1 integrin mAb TS2/16 and IB with JAK2 antibodies (top) or IL-3R β common antibody (bottom). (E) Cell extracts from endothelial cells with (+) or without (−) IL-3 were IP with JAK2 antibodies and subjected to SDS-PAGE. Filters were IB with anti-PY antibodies or JAK2 antibodies. The results are representative of three independent experiments.
Mentions: We previously demonstrated that integrin-mediated adhesion triggered JAK2 activation (Brizzi et al., 1999). Here, we investigated the role of JAK2 in the formation of the β1 integrin–IL-3R β common–STAT5A complex (Fig. 2 C). As positive control for the inhibitory effect of the JAK2 inhibitor AG-490, M07-E cells were used (Fig. 4 A). Subsequently, endothelial cells pretreated with 100 nM were kept in suspension or plated on FN and cell extracts were IP with the antiserum to the IL-3R β common. The anti–phospho-STAT5 immunoblot in Fig. 4 B shows that the phosphorylated STAT5A did not coimmunoprecipitate with the IL-3R β common in adherent cells pretreated with AG-490. In addition, the finding that STAT5A cannot be detected in the IL-3R β common immunoprecipitate from cells pretreated with AG-490 sustains the possibility that JAK2 kinase activity regulates both STAT5 recruitment and activation (Fig. 4 B). Similarly, in the immunoprecipitates of the IL-3R β common, the phosphorylated bands corresponding to STAT5A and the IL-3R β common were not present when the cells were pretreated with AG490 (Fig. 4 C). Moreover, by immunoprecipitating with antibodies to the β1 integrin, JAK2 was found associated with the β1 integrin–IL-3R β common complex only in cells adherent to FN (Fig. 4 D). Adhesion to FN also triggers JAK2 tyrosine phosphorylation (Fig. 4 E). These data support a potential role of JAK2 in regulating both adhesion-mediated IL-3R β common phosphorylation and STAT5A recruitment and activation.

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