NFluc-FHA2-Aktpep-CFluc
Huiming Zhang
PMID: 20641681
Abstract:
The serine/threonine kinase (Akt), also known as protein kinase B (PKB), is an enzyme that covalently attaches ATP-phosphate groups to the serine/threonine on protein substrates to alter the activity of the targeted protein (1, 2). As the human homolog of the viral oncogene v-akt, Akt has three isoforms derived for distinct genes: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ (3). All Akts have an NH2-terminal pleckstrin homology (PH) domain with an activating phosphorylation site (Thr-308) followed by a kinase domain, and a short COOH-terminal regulatory tail with an activating phosphorylation site (Ser-473) (4). The activation of Akt is initiated with the plasma membrane recruitment of phosphatidylinositol 3-kinase (PI3K), an upstream enzyme stimulated by a variety of activated growth factor receptors such as epidermal growth factor receptor (EGFR), vascular growth factor receptor (VEGFR), basic fibroblast growth factor (bFGF), and insulin-like growth factor receptor (IGFR) to generate phospholipids (phosphatidylinositol 3,4,5-triphosphate (PIP3)) to regulate Akt activity (4). The activation of Akt is completed via the phosphorylation of Thr-308 and Ser-473 (i.e., in Akt1), leading to increased Akt activity toward a variety of downstream substrates, such as the family of forkhead transcription factors for inhibiting tumor proliferations, the mammalian target of rapamycin (mTOR) for modulating protein synthesis, and the Bcl-xL/Bcl-2-associated death promoter (BAD) for uncontrolled proliferation (3, 5). These substrates possess a consensus motif R-X-R-XX-ST-B (X = amino acid, B = bulky hydrophobic residue) for Akt phosphorylation (6). Because Akt acts as a signal hub in the regulation of cell survival, proliferation, and growth, the elevation in Akt activity is found to be correlated with the increased tumorigenicity (1). Thus, a variety of inhibitors targeting Akt or its up- or downstream events are currently under clinical trials (7). For example, PAI-2 (triciribine) and perifosine are potent and selective inhibitors of Akt, rapamycin is an inhibitor for downstream mTOR, and LY294002 is an inhibitor for upstream PI3K. Also, molecular imaging of Akt activity has become an important approach in monitoring Akt activity in vivo.
Firefly luciferase (Fluc) is an oxygenase extracted from Photinus pyralis and has a molecular weight of 62 kDa (8). In the presence of adenosine triphosphate (ATP) and O2, Fluc oxidizes the heterocyclic substrate d-luciferin to oxyluciferin and emits light in the wavelength range of 400-620 nm (9). The active site of Fluc is composed of two distinct domains, a large N-terminal domain (residue 4-436) and a small C-terminal domain (residue 440-544), which are separated by a wide cleft (8). Splitting Fluc into N- and C-terminal fragments destroys its enzymatic activity, resulting in a complete loss of bioluminescence. The enzymatic activity or bioluminescence can be restored if the N- and C-terminal fragments are in close proximity (10). This led to the development of the split reporter, a novel labeling strategy for imaging protein-protein interactions in vivo (11). In this method, reporters like Fluc are dissected into two fragments and fused to a pair of proteins (A and B) that strongly interact with each other. The enzymatic activity of Fluc can be restored via a complementation strategy. In this strategy, protein A is connected with the N-terminal fragment of Fluc, and protein B is connected with the C-terminal fragment of Fluc. Interaction between protein A and B recovers the enzymatic activity of Fluc by bringing the two fragments of Fluc closely together, which allows for recovery of bioluminescence.
NFluc-FHA2-Aktpep-CFluc (BARNCFluc) is an optical agent for imaging Akt activity in vivo (2). BARNCFluc consists of four sequentially linked components: an N-terminal Fluc (N-Fluc) fragment, a forkhead-associated (FHA) domain extracted from Rad53 protein kinase (FHA2), an Akt consensus substrate peptide (QSRPRSCTWPLPRPEPRKKK) with spacer linkers (GGSGG) at both ends (Aktpep), and a C-terminal Fluc (C-Fluc). FHA2 (65 amino acids) possesses a β-sandwich that contains two antiparallel β-sheets and a short C-terminal α-helix, and it is able to recognize phosphoproteins by binding to phospho-amino acids such as phospho-Ser and phosphor-Thr (12). The phosphorylation of the Akt consensus substrate peptide in BARNCFluc provides such a target for FHA2 (2). The binding of FHA2 to the phosphorylated Ser/Thr generates steric hindrance to the intramolecular complementation of the N-Fluc and the C-Fluc fragments, leading to a split pair of N/C-Fluc fragments and loss of its bioluminescence. The loss of bioluminescence is directly proportional to the Akt activity; that is, the inhibition of Akt corresponds to enhanced bioluminescence. Thus, BARNCFluc is suitable for monitoring Akt activity in vivo.
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