Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

AbstractInsulin impairs β2-adrenergic receptor (β2AR) function through G protein-coupled receptor kinase 2 (GRK2) by phosphorylation but less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A). Pharmacologic or genetic inhibition of phosphoinositide 3-kinase γ (PI3Kγ) unexpectedly resulted in significant reduction of insulin-mediated β2AR phosphorylation. Interestingly, β2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3Kγ showing negative regulation of PP2A by PI3Kγ. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3Kγ form a complex and could be recruited to β2ARs as GRK2 interacts with insulin receptor substrate following insulin treatment. Consistently, β-blocker pretreatment did not reduce insulin-mediated β2AR phosphorylation indicating agonist- and Gβγ-independent non-canonical regulation of receptor function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating β2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-β2AR cross-talk.SummaryInsulin impairs β2-adrenergic receptor (β2AR) function through G protein-coupled receptor kinase 2 (GRK2). We show that insulin simultaneously inhibits protein phosphatase 2A (PP2A) sustaining β2AR functional impairment. Unexpectedly, releasing PP2A inhibition by PI3Kγ preserves β2AR function despite intact insulin-driven GRK2-mechanisms.

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Abstract P427: Inhibition Of PP2A By Insulin Impairs Beta-Adrenergic Receptor Function

Circulation Research ◽

10.1161/res.129.suppl_1.p427 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Anita Sahu ◽

Sromona D Mukherjee ◽

Conner P Witherow ◽

Kate Stenson ◽

John Tesmer ◽

...

Keyword(s):

Cross Talk ◽

Adrenergic Receptor ◽

Receptor Kinase ◽

Receptor Function ◽

Knock Down ◽

Phosphatase 2A ◽

Β2 Adrenergic Receptor ◽

Β Blocker ◽

Phosphoinositide 3 Kinase ◽

G Protein Coupled

Insulin impairs β2-adrenergic receptor (β2AR) function via trans-phosphorylation through G protein-coupled receptor kinase 2 (GRK2). However, less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A) during this insulin-β2AR cross-talk. Pharmacologic or genetic inhibition of phosphoinositide 3-kinase γ (PI3Kγ) unexpectedly resulted in significant reduction of insulin-mediated β2AR phosphorylation. Interestingly, β2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3Kγ showing negative regulation of PP2A by PI3Kγ. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3Kγ form a complex and could be recruited to β2ARs as GRK2 interacts with insulin receptor substrate (IRS) following insulin treatment. Further, co-immunoprecipitation studies showed that PI3Kγ directly interacted with both IRS-1 and IRS-2 but only IRS-2 interaction with PI3Kγ significantly increased following insulin stimulation. These results indicated that PI3Kγ could also be directly recruited to the receptor complex by IRS-2. Consistently, β-blocker pretreatment did not reduce insulin-mediated β2AR phosphorylation indicating agonist- and Gβγ-independent non-canonical regulation of receptor function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating β2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-β2AR cross-talk.

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Abstract 466: Protein Phosphatase 2A and c-Myc as Intracellular Messengers in the Natriuretic Renal Dopaminergic System

Hypertension ◽

10.1161/hyp.62.suppl_1.a466 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Hanh T Tran ◽

John J Gildea ◽

Robin A Felder

Keyword(s):

G Protein ◽

Protein Phosphatase ◽

Sodium Excretion ◽

Protein Phosphatase 2A ◽

Camp Accumulation ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Protein Levels ◽

Phosphatase 2A ◽

G Protein Coupled

G protein-coupled receptor kinase 4 (GRK4) is known to negatively regulate the dopamine-1 receptor (D 1 R) in human renal proximal tubule cells (RPTC) leading to reduced sodium excretion. c-Myc is a transcription factor involved in positive regulation of G protein-coupled receptor kinase 4 (GRK4). Protein phosphatase 2A (PP2A) inhibits c-Myc by dephosphorylating a residue that normally stabilizes c-Myc. We have previously shown that stimulation of the natriuretic D 1 R in RPTC led to an increased ratio of PP2A/c-Myc binding. Treatment with PMA (protein kinase C inhibitor) led to a decreased PP2A/c-Myc ratio and a lack of cAMP accumulation after stimulation with fenoldopam (FEN, D 1 R agonist). We hypothesized that PP2A plays a key role in regulating natriuresis and that perturbation of PP2A would directly have effects on protein levels of c-Myc, the ratio of PP2A/c-Myc, and the accumulation of cAMP. We used normal RPTCs (nRPTC) and RPTCs that have an uncoupled D 1 R that no longer stimulates adenylyl cyclase (uRPTC). Inhibition of PP2A in uRPTCs with okadaic acid (OA, 100nM, 3 hr) caused an increase in c-Myc protein levels (97.8% ± 18.9 SEM; n=6; p<0.05 (1.44 / 0.73 RFU)), a decrease in the PP2A/c-Myc ratio (-81.8% ± 1.5 SEM; n=6; p<0.05 (1.42 /7.82 RFU)), and a lack of cAMP accumulation upon treatment with SKF38393 (a D 1 R agonist similar to FEN). Activation of PP2A with FTY720 (PP2A activator, 10μM, 3hr) caused a decrease in c-Myc protein levels (- 85.4% ± 2.3 SEM; n=6; p<0.005 (0.11/ 0.73 RFU)), an increase in the PP2A/c-Myc binding ratio by 345.3% ± 90.3 SEM; n=6; p<0.01 (34.82/ 7.82 RFU), and an increase in cAMP accumulation upon stimulation with SKF38393 (94.0% ± 12.4 SEM; n=3; p<0.05 (9.04/4.66 pmole cAMP/mg protein) compared to VEH. In summary, the D 1 R coupling defect found in uRPTCs was restored through activation of PP2A and inhibition of c-Myc. We conclude that PP2A interacts with c-Myc to regulate the natriuretic effect of the D 1 R providing additional insight into the intracellular regulatory events surrounding sodium excretion.

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