Development of Akt-activated GSK3β inhibitory peptide

https://doi.org/10.1016/j.bbrc.2013.03.103Get rights and content

Highlights

  • We designed a novel peptide sequence to inhibit GSK3beta.

  • Activation of the peptide requires the kinase activities of Akt and GSK3beta.

  • GSK3beta is inhibited by the peptide only in response to the insulin signaling.

  • We propose a novel concept to develop a drug.

Abstract

Abnormal overexpression of GSK3β has been implicated in insulin resistance. Although many potent GSK3β inhibitors have been developed as drug candidates for anti-insulin resistance, the inhibitors are prone to show side effects because they interfere with normal GSK3β function without regulation. Recently, it was reported that the PPPSPxS motifs in the Wnt coreceptor LRP6 were able to directly inhibit GSK3β only when the motif was phosphorylated. Here, we generated a new GSK3β inhibitory peptide that can be activated by Akt by combining the PPPSPxS motif and an Akt target sequence. The peptide exhibited an inhibitory effect on GSK3β only when it was phosphorylated by Akt in a purified system and in cells when stimulated by insulin. Thus, our findings provide a novel concept for drugs against diseases that are involved in the abnormal GSK3β activity, including type 2 diabetes mellitus.

Introduction

Glycogen synthase kinase 3β (GSK3β) is a serine-threonine kinase that was initially described as a key enzyme for phosphorylating and inhibiting glycogen synthase [1]. GSK3β plays fundamental roles in controlling various cellular functions, including cell division, proliferation, differentiation, and adhesion [2], [3]. In response to insulin, GSK3β activity is suppressed by phosphorylation at Ser9 by Akt through an autoinhibitory mechanism in peripheral tissues [4], [5], [6].

Accumulating results suggest that hyperactivation of GSK3β in cells is associated with many human diseases, including type 2 diabetes [7], [8]. Type 2 diabetes, which accounts for approximately 90% of all diabetes, characteristically begins with insulin resistance [9]. To date, many studies on this have been performed on the molecular mechanism for insulin resistance, although it still remains to be elucidated. As one of possible mechanisms, elevated GSK3β activity has been implicated in insulin resistance. GSK3β protein levels are significantly higher in muscle biopsies from patients with type 2 diabetes, and blood glucose levels are effectively decreased by inhibition of GSK3 in rodent models of type 2 diabetes [10], [11], [12]. This elevation of GSK3β inversely correlates with both glycogen synthase (GS) activity and insulin-stimulated glucose disposal [12]. The hyperactivation of GSK3β is also associated with the ribosomal protein S6 kinase isoform 1 (S6K1), which was implicated in the insulin resistance [13], [14]. A recent study revealed that GSK3β positively regulates S6K1 [15], and thus use of drugs targeting GSK3β to treat type 2 diabetes was suggested. Even if insulin effectively activates Akt, the total GSK3β activity was not sufficiently suppressed by Akt in the muscle of the diabetic subject [16]. Thus these observations indicated that the efficient inhibition of GSK3β by Akt can be an important step to alleviate the insulin resistance.

GSK3β has been chosen as a potential therapeutic target in multiple diseases including type 2 diabetes mellitus [10], [17]. LiCl was reported as the first GSK3β inhibitor in 1996 [18], and more than 30 small molecule GSK3 inhibitors have already been identified including SB216763 and SB415286 [10], [19], [20]. However, none of them has been approved for clinical use [21], probably because these inhibitors are prone to show side effects if they interfere with normal GSK3β functions. To resolve this unwanted side effect, we designed a GSK3β inhibitory peptide that can be activated by Akt in response to insulin stimulation. Our findings provide a new concept for drugs against diseases that are involved in the abnormal GSK3β activity.

Section snippets

Plasmids and antibodies

DNA fragments encoding LRP6 motif C (residues 1565–1586 of human LRP6) were obtained by PCR from a human cDNA library, and inserted into the pGEX-TEV vector (a modified vector from pGEX-4T1) using EcoRI and XhoI sites to express the peptide in Escherichia coli as a GST-fusion protein with a TEV protease cleavage site. DNA fragments encoding EPVPPPPTPRSSRSRTTSFSES (called LRP-GSK) and EPVPPPPAPRSSRSRTTSFSES (called LRP-GSKmt) were obtained from synthesized genes by Bioneer (Daejeon, Korea) [31].

Design of Akt-activated GSK3β inhibitory peptide

We sought potent GSK3β inhibitory sequences, whose inhibitory activity was raised by phosphorylation. The well-known N-terminal region of GSK3β, which contains Ser9, might be a good candidate for this query. However, the inhibitory effect of the sequence was expected to be too weak to inhibit GSK3β when it was added separately (Ki = 700 μM) [22]. Recent findings have revealed that five iterative PPPSPxS motifs in the cytoplasmic domain of the Wnt co-receptor LRP6 act as a direct inhibitor of GSK3β

Discussion

Besides type 2 diabetes, GSK3β is involved in several other diseases, including inflammation, bipolar disorder, Alzheimer’s disease, and other neurodegenerative diseases [29]. In the process of the downregulation of the TLR-mediated inflammation signaling, the downregulation of the proinflammatory signals is retarded when the GSK3β activity is efficiently inhibited by the Akt [30]. Herein we designed a novel peptide by combining the PPPSPxS motif from the LRP6 C-terminal region and the RxRxxS

Acknowledgments

This study was supported by a Grant from the National Research Foundation of Korea to N.C. Ha (2011-0015420). The authors declare that they have no competing financial interests.

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  • Cited by (0)

    1

    These authors equally contributed to this work.

    2

    Current address: Institute of Molecular and Cell Biology, ASTAR, Singapore 138673, Republic of Singapore.

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