Role Of Phosphoinositide-dependent Protein Kinase 1 Research Articles

Abstract 22: Crosstalk Between Phosphotidylinositol-4,5 Bisphosphonate-3 Kinase Pathway and Mitogen-Activated Protein Kinase Pathway Regulates Platelet Activation and Protein Synthesis via Phosphoinositide-Dependent Kinase-1

Phosphoinositide-dependent protein kinase 1 (PDK1) is known to regulate PAR4 induced platelet activation and thrombus formation through GSK3β. However, whether PDK1 signaling also involves the ADP receptor and, if so, downstream functional consequences are unknown. We employed both pharmacologic (e.g. the selective PDK1 inhibitor, BX795) and genetic (platelet specific deletion of PDK1) approaches to dissect the role of PDK1 in ADP-induced platelet activation and protein synthesis. Inhibition of PDK1 with BX795 reduced 2MeSADP-induced platelet aggregation by abolishing thromboxane generation. Similar results were observed in PDK1 -/- mice (Fig A). Inhibition of PDK1 protected mice from collagen and epinephrine-induced pulmonary embolism (Fig B). PDK1 was also necessary for the phosphorylation of MEK1/2, Erk1/2 and cPLA2, indicating that PDK1 regulates an upstream kinase in MAPK pathway. We next identified that this upstream kinase is Raf1 (necessary for the phosphorylation of MEK1/2), as pharmacologic inhibition and genetic ablation of PDK1 was sufficient to prevent Raf1 phosphorylation (Fig C). Pharmacologic inhibition and genetic ablation of PDK1 blocked MAPK- and mTORC1-dependent protein synthesis in platelets through a mechanism requiring the phosphorylation of eIF4E and S6K. Concordantly, PDK1 is necessary for signal-dependent synthesis of the protein bcl3, which is under mTORC1-dependent control (Fig C). Taken together, our findings show for the first time that PDK1, a master kinase in the PI3K pathway, directly governs thromboxane generation, thrombosis, and protein synthesis in platelets through regulating MAPK and mTORC1 pathways.

Abstract 2238: Role of phosphoinositide-dependent protein kinase-1 (PDK1) in leukemogenesis of acute leukemia

Abstract The Phosphoinositide-dependent Protein Kinase-1 (PDK1) is a key regulator of AKT signaling by phosphorylation of a threonine residue at codon 308. PDK1 itself is activated by PI3kinases and plays an important role in growth factor signaling. We previously demonstrated that AKT is globally activated in acute leukemia, e.g. via gain-of function tyrosine kinase mutations. Consecutively we have shown that tyrosine kinase inhibitors suppress codon 473 phosphorylation - but leave Thr308 activated, hypothetically via activation by growth factors. Accordingly, we show that phosphorylation status of AKT in bone marrow leukemia blasts is higher compared to circulating blasts, which favors the hypothesis of a protective bone marrow microenvironment due to the vast occurrence of growth factors and chemokines, which activate PI3K/AKT signaling. We aimed to investigate whether activation of AKT(Thr308) is crucial for the maintenance of leukemia cells and leukemogenesis and silenced PDK1 by retrovirally transducing different short hairpin RNA constructs cloned into a mammalian vector into AML cell lines. Proliferation capacity of the PDK1-silenced cells in comparison to an empty vector control was studied using an XTT-based assay. Cell lines with suppressed transcription of PDK1 revealed a significantly diminished proliferation capacity compared to the empty vector controls. To evaluate potency of tumor engraftment and aggressiveness of disease in vivo, we transplanted stably transfected PDK1-shRNA silenced acute leukemia cells into NOD/SCID mice. Isogenic cells transfected with an empty vector were used as the control group. Mice were regularly monitored for disease symptoms and leukemic blast count. Already on day 8, the peripheral blast count in the control mice revealed a faster increase with higher cell counts (∼ 4 x 10ex3/microL) compared to the PDK1-silenced group (∼ 0,5 x 10ex3/microL). Over time, all mice finally developed leukemia, with the control mice to be the first. In addition, we tested two novel PI3K/mTOR inhibitors, NVP-BEZ235 and NVP-BGT226, with regard to the antiproliferative capacity in ex vivo leukemia blasts. Both agents revealed potent antiproliferation with NVP-BGT226 the more potent agent. NVP-BGT226 induced apoptosis in ex vivo blasts in a subset of patients, with an IC50 in the low nanomolar range. In summary, we here identify the PDK1-AKT signal transduction pathway to be crucial for aggressiveness of disease. Of note, it was not possible to suppress development of leukemia completely, arguing for a multistep mechanism of leukemogenesis. Our data supports descriptive expression analysis of PDK1 or AKT(Thr308) that link high expression with unfavorable clinical outcome. Therefore, targeting the PI3K/AKT axis which activates PDK1 in acute leukemia may be beneficial to improve therapy outcome in a subset of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2238. doi:1538-7445.AM2012-2238

Roles of phosphoinositide-dependent kinase-1 in α1B-adrenoceptor phosphorylation and desensitization

The role of phosphoinositide-dependent protein kinase-1 (PDK-1) activity on α1B-adrenoceptor phosphorylation and function was explored using pharmacological inhibitors and expression of a dominant-negative mutant of this enzyme. Noradrenaline-, phorbol myristate acetate-, lysophosphatidic acid- and epidermal growth factor-mediated α1B-adrenoceptor phosphorylation were markedly reduced by the two inhibitors used: UCN-01 [(7-hydroxystaurosporine; (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one)] and OSU-03012 [(2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide)]. A similar effect was observed in cells expressing a PDK-1 dominant-negative mutant. Phosphorylated PDK-1 (S241) and protein kinase C α (T497) were associated with cell membranes in the basal state which increased in response to the hormonal stimuli mentioned previously. UCN-01 essentially abolished phospho-PDK-1 membrane-association and markedly attenuated that of protein kinase C α. Consistent with the findings, UCN-01 reduced lysophosphatidic acid- and epidermal growth factor-induced α1B-adrenoceptor desensitization. Our data suggest that PDK-1 plays a permissive role in α1B-adrenoceptor desensitization and phosphorylation and participates in the formation of signaling complexes, which delicately modulate receptor function and regulation.

Targeting PDK1 in Cancer

Abnormal activation of phosphoinositide 3-kinase (PI3K) signalling is very common in cancer, leading to deregulation of several intracellular processes normally controlled by this enzyme, including cell survival, growth, proliferation and migration. Mutations in the gene encoding the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which leads to uncontrolled activation of the PI3K pathway, are reported in different cancers. Among the downstream effectors of PI3Ks, 3- phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB)/Akt have a key role in several cancer types. More recent data indicate that alteration of PDK1 is a critical component of oncogenic PI3K signalling in breast cancer, suggesting that inhibition of PDK1 can inhibit breast cancer progression. PDK1 has an essential role in regulating cell migration especially in the context of PTEN deficiency. Downregulation of PDK1 levels inhibits migration and experimental metastasis of human breast cancer cells. PDK1 activates a large number of proteins, including Akt, some PKC isoforms, S6K and SGK. Data also reveal that PDK1 is oncogenic and this is dependent on PI3K pathway. Therefore, accumulating evidence demonstrates that PDK1 is a valid therapeutic target and suggests that PDK1 inhibitors may be useful to prevent cancer progression and abnormal tissue dissemination. This review will focus on published data on the role of PDK1 in cancer and approaches used to inhibit PDK1.