Activation of a GST-tagged AKT2/PKBβ

https://doi.org/10.1016/j.bbagen.2005.04.002Get rights and content

Abstract

The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (ΔPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (kcat/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.

Introduction

The serine/threonine protein kinase B/AKT exists as three highly homologous isoforms, AKT1, AKT2, and AKT3, (PKBα/β/γ, respectively) and has been implicated to be involved in the regulation of cell growth, apoptosis, and insulin action [1], [2]. Upon stimulation with insulin, AKT is recruited to cellular membranes by binding of its amino terminal pleckstrin (PH) domain to membrane bound phosphatidylinositol 3,4,5, trisphosphate (PIP3) [3]. The membrane bound form of AKT then becomes phosphorylated on two regulatory residues, a threonine within the activation loop (Thr308 in AKT1,Thr309 in AKT2, Thr305 in AKT3) and a serine in the C-terminus of the enzyme (Ser473 in AKT1,Ser474 in AKT2, Ser472 in AKT3), and both phosphorylations are considered to be required for AKT to reach maximum kinase activity [1]. The kinase responsible for phosphorylation of Thr308/309/305 has been identified as phosphoinositide-dependent kinase 1 (PDK1) [3]. However, despite intensive experimental efforts in the recent years, identification of the kinase responsible for the phosphorylation of Ser473/474/472 in AKT, tentatively designated PDK2, remains controversial. Several different kinases have been proposed to phosphorylate AKT1's Ser473, including PDK1 [4], Integrin-linked kinase [5], mitogen-activated protein kinase-activated kinase-2 (MAPKAPK-2) [6], [7], a distinct but unidentified kinase associated with cytoskeletal elements of the plasma membranes [8], [9], and most recently DNA-dependent protein kinase (DNA-PK) [10]. Intriguingly, it also has been shown that phosphorylation of AKT1 on Ser473 in vitro occurs as the result from an autophosphorylation event after PDK1-mediated phosphorylation of Thr308 [11]. Moreover, oligomerization of AKT has been proposed to contribute to the activation of the kinase [12], [13]. In particular, oligomerization of AKT (via its PH domain; [14]) mediated by the proto-oncogen TCL1 has been shown to promote trans-phosphorylation of both Ser473/474/472 and Thr308/309/305 in vitro [15]. However, in both studies, autophosphorylation of AKT has only been demonstrated with partially purified enzyme preparations [11], [15].

In order to characterize the AKT2 isoform which is enriched in insulin-responsive tissues [16], we have cloned, expressed, and purified the kinase, and various mutants as glutathione S-transferase (GST) fusion proteins via the baculovirus expression system. Because GST fusion proteins form stable dimers during protein synthesis [17], [18], we were able to compare the catalytic properties of the non-tagged monomeric form with the dimerized GST-AKT2 kinase.

Section snippets

Cloning and expression of GST-AKT2

A cDNA fragment of human AKT2/PKBβ (a generous gift from Dr. Morris J. Birnbaum) corresponding to amino acids Pro119–Glu481 (ΔPH-AKT2) was obtained by NcoI/BglII digestion and Klenow fill-in as described previously [19], and ligated into a SmaI/BamHI digested baculovirus transfer vector pAcG2T (Pharmingen). On the cDNA level, this resulted in fusion of ΔPH-AKT2 to the C-terminus of glutathione-S-transferase to eventually yield a 68-kDa GST fusion protein. Generation of the point mutations of

Expression and purification of GST-AKT2

A cDNA fragment of human AKT2 was subcloned into the baculovirus recombination vector pAcG2T to generate a 68-kDa fusion protein of glutathione-S-transferase and AKT2. The construct included the catalytic domain and the C-terminal domain of AKT2 (amino acids Pro119–Glu481) but lacked AKT's N-terminal pleckstrin homology (PH) domain (Fig. 1A). In addition, we generated two mutants of GST-AKT2, a kinase-inactive form where Lys181 in the ATP-binding site was exchanged for alanine (GST-AKT2K/A),

Discussion

The GST-kinases were purified to homogeneity from insect cells with a yield of 5–15 mg per liter of cell culture. The purified unphosphorylated form of GST-AKT2 displayed significant but low kinase activity (Fig. 2, Fig. 3). As reported previously for AKT1 [26], treatment of the cells with the phosphatase inhibitor okadaic acid led to substantial phosphorylation of both Thr309 and Ser474 (Fig. 2), and to activation of GST-AKT2 (Fig. 3, Fig. 4). Compared to the control, the maximum velocity of

Acknowledgements

We thank Dr. Gursant S. Kular for providing the purified PDK1. This work was funded in part by the Deutsche Diabetes Gesellschaft.

References (34)

Cited by (0)

1

Present address: EDMNS/DB/NIDDK, National Institutes of Health, Bethesda, MD, USA.

View full text