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Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13.

Bentin Toaldo C, Alexi X, Beelen K, Kok M, Hauptmann M, Jansen M, Berns E, Neefjes J, Linn S, Michalides R, Zwart W - BMC Cancer (2015)

Bottom Line: Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13.Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. c.bentin.toaldo@nki.nl.

ABSTRACT

Background: Estrogen Receptor alpha (ERα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.

Methods: We performed immunohistochemistry to detect ERαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 K gene expression analyses were performed and integrated with clinicopathological data and survival information. In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERαSerine 305 phosphorylation status, ERα/PKA interactions and downstream responsive gene activity.

Results: Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.

Conclusions: We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients.

No MeSH data available.


Related in: MedlinePlus

Model for PKA-mediated ERαS305 phosphorylation and the role of AKAP13 in this process. AKAP13 interacts with ERα as well as PKA-RII. This way, AKAP13 functions as a scaffolding protein, bringing together the PKA complex and its substrate, ERα. When members of the seven-transmembrane domain (7TM) receptors/ G protein-coupled receptors (GPCRs) are activated, this results in an activation of Adenyl Cyclase to generate cAMP. PKA is consequently activated, by the association of the PKA-regulatory subunits with cAMP. Subsequently, the catalytic subunit can dissociate from the complex and directly phosphorylate ERα at Serine 305. This phosphorylation at ERαS305P results in the recognition of tamoxifen as an ERα agonist, initiation of tamoxifen-driven gene transcription and consequently tamoxifen resistance
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Fig5: Model for PKA-mediated ERαS305 phosphorylation and the role of AKAP13 in this process. AKAP13 interacts with ERα as well as PKA-RII. This way, AKAP13 functions as a scaffolding protein, bringing together the PKA complex and its substrate, ERα. When members of the seven-transmembrane domain (7TM) receptors/ G protein-coupled receptors (GPCRs) are activated, this results in an activation of Adenyl Cyclase to generate cAMP. PKA is consequently activated, by the association of the PKA-regulatory subunits with cAMP. Subsequently, the catalytic subunit can dissociate from the complex and directly phosphorylate ERα at Serine 305. This phosphorylation at ERαS305P results in the recognition of tamoxifen as an ERα agonist, initiation of tamoxifen-driven gene transcription and consequently tamoxifen resistance

Mentions: As a model for PKA-mediated ERαS305P phosphorylation, leading to tamoxifen resistance, we propose the following order of events (as illustrated in Fig. 5): AKAP13 interacts with ERα as well with PKA-RII. PKA-RII forms a complex with PKA-RI and the catalytic subunit of PKA, so that the entire PKA complex is indirectly associated to ERα. This interaction is present in the absence of PKA activation. When cAMP levels increase, cAMP can bind PKA-RI and PKA-RII, so that PKA-cat is dissociated from the complex. By virtue of the preformed protein complex that is mediated by AKAP13, the activated PKA-cat can locally act and phosphorylate ERαS305. When the phosphorylated ERα binds tamoxifen, ERα is selectively targeted to a distinct set of gene promoters to activate these genes, resulting in tamoxifen resistance [13].Fig. 5


Protein Kinase A-induced tamoxifen resistance is mediated by anchoring protein AKAP13.

Bentin Toaldo C, Alexi X, Beelen K, Kok M, Hauptmann M, Jansen M, Berns E, Neefjes J, Linn S, Michalides R, Zwart W - BMC Cancer (2015)

Model for PKA-mediated ERαS305 phosphorylation and the role of AKAP13 in this process. AKAP13 interacts with ERα as well as PKA-RII. This way, AKAP13 functions as a scaffolding protein, bringing together the PKA complex and its substrate, ERα. When members of the seven-transmembrane domain (7TM) receptors/ G protein-coupled receptors (GPCRs) are activated, this results in an activation of Adenyl Cyclase to generate cAMP. PKA is consequently activated, by the association of the PKA-regulatory subunits with cAMP. Subsequently, the catalytic subunit can dissociate from the complex and directly phosphorylate ERα at Serine 305. This phosphorylation at ERαS305P results in the recognition of tamoxifen as an ERα agonist, initiation of tamoxifen-driven gene transcription and consequently tamoxifen resistance
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536754&req=5

Fig5: Model for PKA-mediated ERαS305 phosphorylation and the role of AKAP13 in this process. AKAP13 interacts with ERα as well as PKA-RII. This way, AKAP13 functions as a scaffolding protein, bringing together the PKA complex and its substrate, ERα. When members of the seven-transmembrane domain (7TM) receptors/ G protein-coupled receptors (GPCRs) are activated, this results in an activation of Adenyl Cyclase to generate cAMP. PKA is consequently activated, by the association of the PKA-regulatory subunits with cAMP. Subsequently, the catalytic subunit can dissociate from the complex and directly phosphorylate ERα at Serine 305. This phosphorylation at ERαS305P results in the recognition of tamoxifen as an ERα agonist, initiation of tamoxifen-driven gene transcription and consequently tamoxifen resistance
Mentions: As a model for PKA-mediated ERαS305P phosphorylation, leading to tamoxifen resistance, we propose the following order of events (as illustrated in Fig. 5): AKAP13 interacts with ERα as well with PKA-RII. PKA-RII forms a complex with PKA-RI and the catalytic subunit of PKA, so that the entire PKA complex is indirectly associated to ERα. This interaction is present in the absence of PKA activation. When cAMP levels increase, cAMP can bind PKA-RI and PKA-RII, so that PKA-cat is dissociated from the complex. By virtue of the preformed protein complex that is mediated by AKAP13, the activated PKA-cat can locally act and phosphorylate ERαS305. When the phosphorylated ERα binds tamoxifen, ERα is selectively targeted to a distinct set of gene promoters to activate these genes, resulting in tamoxifen resistance [13].Fig. 5

Bottom Line: Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13.Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, the Netherlands Cancer Institute, Amsterdam, The Netherlands. c.bentin.toaldo@nki.nl.

ABSTRACT

Background: Estrogen Receptor alpha (ERα)-positive breast cancer patients receive endocrine therapy, often in the form of tamoxifen. However, resistance to tamoxifen is frequently observed. A signalling cascade that leads to tamoxifen resistance is dictated by activation of the Protein Kinase A (PKA) pathway, which leads to phosphorylation of ERα on Serine 305 and receptor activation, following tamoxifen binding. Thus far, it remains elusive what protein complexes enable the PKA-ERα interaction resulting in ERα Serine 305 phosphorylation.

Methods: We performed immunohistochemistry to detect ERαSerine 305 phosphorylation in a cohort of breast cancer patients who received tamoxifen treatment in the metastatic setting. From the same tumor specimens, Agilent 44 K gene expression analyses were performed and integrated with clinicopathological data and survival information. In vitro analyses were performed using MCF7 breast cancer cells, which included immunoprecipitations and Fluorescence Resonance Energy Transfer (FRET) analyses to illustrate ERα complex formation. siRNA mediated knockdown experiments were performed to assess effects on ERαSerine 305 phosphorylation status, ERα/PKA interactions and downstream responsive gene activity.

Results: Stratifying breast tumors on ERα Serine 305 phosphorylation status resulted in the identification of a gene network centered upon AKAP13. AKAP13 mRNA expression levels correlate with poor outcome in patients who received tamoxifen treatment in the metastatic setting. In addition, AKAP13 mRNA levels correlate with ERαSerine 305 phosphorylation in breast tumor samples, suggesting a functional connection between these two events. In a luminal breast cancer cell line, AKAP13 was found to interact with ERα as well as with a regulatory subunit of PKA. Knocking down of AKAP13 prevented PKA-mediated Serine 305 phosphorylation of ERα and abrogated PKA-driven tamoxifen resistance, illustrating that AKAP13 is an essential protein in this process.

Conclusions: We show that the PKA-anchoring protein AKAP13 is essential for the phosphorylation of ERαS305, which leads to tamoxifen resistance both in cell lines and tamoxifen-treated breast cancer patients.

No MeSH data available.


Related in: MedlinePlus