Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Treatment with sodium (S)-2-hydroxyglutarate prevents liver injury in an ischemia-reperfusion model in female Wistar rats.

BACKGROUND: Ischemia-reperfusion (IR) injury is one of the leading causes of early graft dysfunction in liver transplantation. Techniques such as ischemic preconditioning protect the graft through the activation of the hypoxia-inducible factors (HIF), which are downregulated by the EGLN family of prolyl-4-hydroxylases, a potential biological target for the development of strategies based on pharmacological preconditioning. For that reason, this study aims to evaluate the effect of the EGLN inhibitor sodium (S)-2-hydroxyglutarate [(S)-2HG] on liver IR injury in Wistar rats. METHODS: Twenty-eight female Wistar rats were divided into the following groups: sham (SH, n = 7), non-toxicity (HGTox, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days), IR (n = 7, total liver ischemia: 20 minutes, reperfusion: 60 minutes), and (S)-2HG+IR (HGIR, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days, total liver ischemia as the IR group). Serum ALT, AST, LDH, ALP, glucose, and total bilirubin were assessed. The concentrations of IL-1ß, IL-6, TNF, malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured in liver tissue, as well as the expression of Hmox1, Vegfa, and Pdk1, determined by RT-qPCR. Sections of liver tissue were evaluated histologically, assessing the severity of necrosis, sinusoidal congestion, and cytoplasmatic vacuolization. RESULTS: The administration of (S)-2HG did not cause any alteration in the assessed biochemical markers compared to SH. Preconditioning with (S)-2HG significantly ameliorated IR injury in the HGIR group, decreasing the serum activities of ALT, AST, and LDH, and the tissue concentrations of IL-1ß and IL-6 compared to the IR group. IR injury decreased serum glucose compared to SH. There were no differences in the other biomarkers assessed. The treatment with (S)-2HG tended to decrease the severity of hepatocyte necrosis and sinusoidal congestion compared to the IR group. The administration of (S)-2HG did not affect the expression of Hmox1 but decreased the expression of both Vegfa and Pdk1 compared to the SH group, suggesting that the HIF-1 pathway is not involved in its mechanism of hepatoprotection. In conclusion, (S)-2HG showed a hepatoprotective effect, decreasing the levels of liver injury and inflammation biomarkers, without evidence of the involvement of the HIF-1 pathway. No hepatotoxic effect was observed at the tested dose.

MicroRNA-4290 suppresses PDK1-mediated glycolysis to enhance the sensitivity of gastric cancer cell to cisplatin

The development of chemotherapy resistance significantly impairs the efficiency of chemotherapy, but the underlying mechanisms of chemotherapy resistance in gastric cancer (GC) are complicated and still need to be further explored. Here, we aimed to reveal the effects of miR-4290/PDK1 (pyruvate dehydrogenase kinase 1) axis on chemotherapy resistance of GC in vitro. The expression patterns of miR-4290 in GC tissues and cell lines were determined by real-time quantitative PCR. Kaplan-Meier was used to assess the relationship between miR-4290 expression levels and patients' overall survival. CCK-8 and flow cytometry technologies were applied to detect cell proliferation and apoptosis. The luciferase gene reporter assay was used to evaluate the interaction between miR-4290 and PDK1. miR-4290 was lowly expressed in GC tissues and cell lines, which was closely associated with the shorter overall survival of GC patients. miR-4290 mimics significantly inhibited cell proliferation and induced cell apoptosis, as well as induced a significant reduction in the expression of PDK1. Moreover, miR-4290 significantly inhibited glycolysis and decreased the IC50 value to cisplatin in SGC7901 cells, whereas these effects were abolished and cell apoptosis was promoted when PDK1 was overexpressed. In conclusion, this study revealed that miR-4290 suppressed PDK1-mediated glycolysis to enhance the sensitivity of GC cells to cisplatin.

Allosteric Regulation of Protein Kinases Downstream of PI3-Kinase Signalling.

Allostery is a basic principle that enables proteins to process and transmit cellular information. Protein kinases evolved allosteric mechanisms to transduce cellular signals to downstream signalling components or effector molecules. Protein kinases catalyse the transfer of the terminal phosphate from ATP to protein substrates upon specific stimuli. Protein kinases are targets for the development of small molecule inhibitors for the treatment of human diseases. Drug development has focussed on ATP-binding site, while there is increase interest in the development of drugs targeting alternative sites, i.e. allosteric sites. Here, we review the mechanism of regulation of protein kinases, which often involve the allosteric modulation of the ATP-binding site, enhancing or inhibiting activity. We exemplify the molecular mechanism of allostery in protein kinases downstream of PI3-kinase signalling with a focus on phosphoinositide-dependent protein kinase 1 (PDK1), a model kinase where small compounds can allosterically modulate the conformation of the kinase bidirectionally.

Expression of PDK1 in malignant pheochromocytoma as a new promising potential therapeutic target.

PURPOSE: Phosphoinositide-dependent kinase 1 (PDK1) is highly expressed in many solid tumors. And several studies have demonstrated that PDK1 has been an emerging and promising target for anti-cancer therapies. However, the role of PDK1 has not been studied so far in malignant pheochromocytoma (PCC). METHODS: In this study, immunohistochemical staining was performed to investigate the protein level of PDK1 in 63 PCC tissue samples, of which 49 were benign and 14 were malignant. In addition, we evaluated the effect of inhibition of PDK1 with siRNA on cell growth, apoptosis and invasive capacity in PC12 cells and identified the underlying mechanisms. RESULTS: We found that PDK1 was overexpressed in malignant PCC tissues, and knockdown of PDK1 with siRNA significantly inhibited cell proliferation, increased apoptosis induction, and attenuated cell migration and invasive capacity in PC12 cells. We also showed that knockdown of PDK1 significantly reduced the phosphorylation of Akt at threonine 308 (p-Akt T308) but did not alter the serine phosphorylation of Akt on the S473 site (p-Akt S473). Furthermore, we found that the p-Akt expression was noticeably decreased after knockdown of PDK1, but the t-Akt expression did not show a significant decrease. CONCLUSION: We have demonstrated for the first time that PDK1 is overexpressed in human malignant PCC and plays an important role in the malignant biological behaviors of PC12 cell. Specifically, we have revealed that knockdown of PDK1 could attenuate activation of the Akt signaling. These data suggest that PDK1 could be a new promising potential therapeutic target in human cancer treatment for malignant PCC.

STAT3 enhances the constitutive activity of AGC kinases in melanoma by transactivating PDK1.

BACKGROUND: The PI3K/Akt and the STAT3 pathways are functionally associated in many tumor types. Both in vitro and in vivo studies have revealed that either biochemical or genetic manipulation of the STAT3 pathway activity induce changes in the same direction in Akt activity. However, the implicated mechanism has been poorly characterized. Our goal was to characterize the precise mechanism linking STAT3 with the activity of Akt and other AGC kinases in cancer using melanoma cells as a model. RESULTS: We show that active STAT3 is constitutively bound to the PDK1 promoter and positively regulate PDK1 transcription through two STAT3 responsive elements. Transduction of WM9 and UACC903 melanoma cells with STAT3-small hairpin RNA decreased both PDK1 mRNA and protein levels. STAT3 knockdown also induced a decrease of the phosphorylation of AGC kinases Akt, PKC, and SGK. The inhibitory effect of STAT3 silencing on Akt phosphorylation was restored by HA-PDK1. Along this line, HA-PDK1 expression significantly blocked the cell death induced by dacarbazine plus STAT3 knockdown. This effect might be mediated by Bcl2 proteins since HA-PDK1 rescued Bcl2, Bcl-XL, and Mcl1 levels that were down-regulated upon STAT3 silencing. CONCLUSIONS: We show that PDK1 is a transcriptional target of STAT3, linking STAT3 pathway with AGC kinases activity in melanoma. These data provide further rationale for the ongoing effort to therapeutically target STAT3 and PDK1 in melanoma and, possibly, other malignancies.

Timp1 Promotes Cell Survival by Activating the PDK1 Signaling Pathway in Melanoma.

High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and ß1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCßIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.

Therapy with radio-attenuated vaccine in experimental murine visceral leishmaniasis showed enhanced T cell and inducible nitric oxide synthase levels, suppressed tumor growth factor-beta production with higher expression of some signaling molecules

Background: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. Methods: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-β) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. Results: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-β level were found in treated infected animal groups (100 and 150 Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. Conclusion: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-β expression has augmented the restored Th1 ambience in the 100 and 150 Gy treated animal groups proving further the efficacy of the candidate vaccine. .

Therapy with radio-attenuated vaccine in experimental murine visceral leishmaniasis showed enhanced T cell and inducible nitric oxide synthase levels, suppressed tumor growth factor-beta production with higher expression of some signaling molecules.

BACKGROUND: Visceral leishmaniasis (VL) or Kala-Azar (KA) is one of the most deadly forms of disease among all neglected tropical diseases. There are no satisfactory drugs or vaccine candidates available for this dreaded disease. Our previous studies showed promising therapeutic and prophylactic efficacy of the live, radio-attenuated parasites through intramuscular (I.M.) and intraperitoneal (I.P.) route in BALB/c mice model. METHODS: The T-cell proliferation level, the mRNA expression level of inducible nitric oxide synthase (iNOS) and tumor growth factor-beta (TGF-ß) genes and finally the phosphorylation levels of phosphoinositide dependent kinase 1 (PDK1), phosphoinositide 3 kinase (PI3K) and p38 mitogen activated protein kinase (p38MAPK) molecules were checked in BALB/c mice model immunized with radio-attenuated Leishmania donovani parasites through I.M. route. RESULTS: Higher T-cell proliferation, increased iNOS level, and suppressed TGF-ß level were found in treated infected animal groups (100 and 150Gy) in relation to untreated infected animals. Likewise, phosphorylation levels of PDK1, PI3K and p38MAPK of these two groups were increased when compared to untreated infected controls. CONCLUSION: The clearance of the parasites from treated infected groups of animals may be mediated by the restoration of T-cell due to therapy with radio-attenuated L. donovani parasites. The killing of parasites was mediated by increase in nitric oxide release through PDK1, PI3K and p38MAPK signaling pathways. A lower TGF-ß expression has augmented the restored Th1 ambience in the 100 and 150Gy treated animal groups proving further the efficacy of the candidate vaccine.

DHEA supplementation in ovariectomized rats reduces impaired glucose-stimulated insulin secretion induced by a high-fat diet.

Dehydroepiandrosterone (DHEA) and the dehydroepiandrosterone sulfate (DHEA-S) are steroids produced mainly by the adrenal cortex. There is evidence from both human and animal models suggesting beneficial effects of these steroids for obesity, diabetes mellitus, hypertension, and osteoporosis, conditions associated with the post-menopausal period. Accordingly, we hypothesized that DHEA supplementation in ovariectomized (OVX) female rats fed a high-fat diet would maintain glucose-induced insulin secretion (GSIS) and pancreatic islet function. OVX resulted in a 30% enlargement of the pancreatic islets area compared to the control rats, which was accompanied by a 50% reduction in the phosphorylation of AKT protein in the pancreatic islets. However, a short-term high-fat diet induced insulin resistance, accompanied by impaired GSIS in isolated pancreatic islets. These effects were reversed by DHEA treatment, with improved insulin sensitivity to levels similar to the control group, and with increased serine phosphorylation of the AKT protein. These data confirm the protective effect of DHEA on the endocrine pancreas in a situation of diet-induced overweight and low estrogen concentrations, a phenotype similar to that of the post-menopausal period.