Insulin induces phosphorylation of serine residues of translationally controlled tumor protein in 293T cells.

Jeehye Maeng,Kyunglim Lee,Miyoung Kim,Hyukjin Lee

doi:10.3390/ijms16047565

Jeehye Maeng, Kyunglim Lee + Show 2 more

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https://doi.org/10.3390/ijms16047565

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Abstract

Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

Highlights

Insulin is known to activate Na,K-ATPase [1] and causes vasodilation via sodium pump activation [2]
We examined whether insulin treatment affects the localization of translationally controlled tumor protein (TCTP) by detecting the p-Ser-TCTP in cytosol and plasma membrane of the transfected 293T cells treated with insulin
We propose that TCTP phosphorylation by insulin may be the stimulus that induces TCTP to translocate from membrane to cytosol and the contributory mechanism for sodium pump regulation

Summary

Introduction

Insulin is known to activate Na,K-ATPase [1] and causes vasodilation via sodium pump activation [2]. Investigations on how insulin stimulates the sodium pump may help to understand the pathophysiology of diabetes and diabetes-induced hypertension. It has been proposed that insulin-induced sodium pump regulation occurs via a still unknown mechanism, involving an as yet unknown cytoplasmic repressor [11]. TCTP inhibits the pump activity of Na,K-ATPase by binding to third cytoplasmic domain of the enzyme’s α subunit [13], resulting in the development of systemic hypertension in TCTP-overexpressing transgenic mice (TCTP-TG) [14]. Inhibition of the Na,K-ATPase activity by TCTP overexpression is responsible for the hypercontractile reactivity of TCTP-TG mice [14]

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