Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

Abstract

Abstract3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C(AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1 −/− ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1 −/− ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.

The PDK1 master kinase is over-expressed in acute myeloid leukemia and promotes PKC-mediated survival of leukemic blasts

“…As predicted from previous studies 19,32 BX-795 was able to inhibit the phosphorylation of PKC and S6 in these patients ( Figure 4C). Inhibition of AKT phosphorylation was also observed, but only at high dose.…”

“…)cells. 19 Consistent with these observations, PDK1 overexpression is a common feature of a wide variety of cancers [20][21][22][23] and an RNAi screen has identified PDK1 as being the most important factor in mediating resistance to tamoxifen in breast cancer. 24 An essential role for PDK1 has also recently been identified in pancreatic cancer.…”

PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

“…For example, the indolinone-based PDK1 inhibitor BX-795 and its analogues inhibit cell cycle progression, induce apoptosis, and show efficacy in a xenograft model (18). However, BX-795 causes G2/M cell cycle arrest in both PDK1 wild-type (WT) and PDK1 knockout cells suggesting that the effects on cell cycle are unrelated to PDK1 inhibition (19). At present, analysis of small-molecule PDK1 inhibitors, covering a dozen structural classes (11), has not pinpointed any selective compound suitable for in vivo efficacy studies.…”

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells

“…However, preclinical efficacy studies using pharmacological inhibitors of PDK1 have been hampered by the lack of specific proof-of-concept molecules (11,13). Thus, gene silencing and expression of functionally impaired and dominant negative mutant forms of PDK1 have frequently been used to probe PDK1 protein function in cells (13)(14)(15)(16)(17)(18)(19)(20)(21).…”