Differentially activating the oncogenic kinase Akt1

The proto-oncogene Akt/protein kinase B plays a pivotal role in cell growth and survival. Phosphorylation of Akt at Thr308 and Ser473 activates the kinase following growth factor stimulation. Delineating specific role of each activation site in Akt1 on kinase activation, inhibition and substrate selection remain elusive. We designed a unique set of tools, relying on genetic code expansion with phosphoserine and in vivo enzymatic phosphorylation, to produce differentially phosphorylated Akt1 variants. We found that having both sites phosphorylated increased the apparent catalytic rate of the enzyme by 1500-fold relative to the unphosphorylated enzyme. This increment was mainly due to the phosphorylation of Thr308 but not Ser473 which was confirmed via live cell imaging. We further found that the traditional use of phosphomimetics was unable to mimic the effect of p-Thr308 in the test tube and in cells. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. Using the same strategy, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three Akt1 phospho-variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. The robustly active PH domain deleted enzyme variants were used in enzyme inhibition and substrate selectivity studies. We found that phosphorylation at Ser473 provided resistance to chemical inhibition by the Akt inhibitor Akti-1/2. Finally, we used both defined and randomized peptide libraries to map the substrate selectivity of singly and doubly phosphorylated Akt1 variants. The data revealed that each phospho-form of Akt1 has distinct substrate requirements. Surprisingly, phosphorylation of Ser473 in the context of a pAkt1T308 enzyme led to increased activity on some, but not all Akt1 substrates. We also verified a new Akt1 target as the terminal nucleotidyltransferase (germline development 2) Gld2. In conclusion, the site-specifically phosphorylated Akt1 variants that we produced enabled in characterizing phosphorylation-dependent activity, inhibition and substrate selectivity of the oncogenic kinase Akt1. Since phosphorylation status of Akt1 is used as a cancer biomarker, these variants can act as indispensable tools in further characterizing downstream oncogenic pathways and screening potential drug candidates

Phosphorylation-dependent inhibition of Akt1

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1

Gld2 activity is regulated by phosphorylation in the N-terminal domain

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling

Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1)

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ~1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes

Phosphorylation-Dependent Inhibition of Akt1

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1

Delivery of Active AKT1 to Human Cells

Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Thr308, Ser473), yet cell stimulation also activates many other kinases. To produce cells with specific AKT1 activity, we developed a novel system to deliver active AKT1 to human cells. We recently established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. Here, we fused AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) protein. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308 induced selective phosphorylation of the known AKT1 substrate GSK-3α, but not GSK-3β, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Ser240/244. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on AKT1 activity

Antihypertensive effects of apple peel extract on spontaneously hypertensive rats

Background and Objectives: Apple peel is a rich source of biological active phytochemicals such as flavonoids. The present study investigated the antihypertensive effect of flavonoid-rich Apple Peel Extract (APE) on Spontaneously Hypertensive Rats (SHR). Methodology: Three groups of animals: Control, captopril (20 mg kg–1 of body weight/day) and APE (25 mg kg–1 of body weight/day) were fed standard rat chow and their corresponding treatment in sugar-free gelatin, daily for a period of eight weeks. Blood Pressure (BP) was monitored weekly using the tail cuff method. Blood and tissue samples were collected after the eighth week. Results: As expected, treatment with captopril consistently reduced BP (p<0.05). APE treatment reduced both systolic and diastolic BP by 15 and 11 mg Hg, respectively, after 5 weeks of treatment, However, statistical significance was only achieved in systolic BP after eight weeks when compared with control (p<0.05). There were no significant differences in serum and lung ACE activity at week eight. Treatment with APE increased liver superoxide dismutase (SOD) activity by 78% and total reduced Glutathione (GSH) concentrations by 42% when compared to control (p<0.05) but had no effect on the activity of glutathione reductase or peroxidase. Conclusion: Long term intake of APE reduces high blood pressure in SHR possibly through endogenous antioxidant pathways. This preclinical trial suggests that APE as a dietary supplement could be effective in managing early stages of hypertension. </p

miRNA-Dependent Regulation of AKT1 Phosphorylation

The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling