The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated
via
protein–protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR–14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR–14-3-3 axis as potential targets for future therapeutic intervention.
The 14-3-3/c-Abl
protein–protein interaction (PPI) is related
to carcinogenesis and in particular to pathogenesis of chronic myeloid
leukemia (CML). Previous studies have demonstrated that molecules
able to disrupt this interaction improve the nuclear translocation
of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography
screening program, we have identified two phosphate-containing compounds,
inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders
of human 14-3-3σ, by targeting the protein amphipathic groove.
Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl
PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library
of PLP and IMP analogues was investigated using a FP assay, leading
to the identification of three further molecules acting as weak inhibitors
of the 14-3-3/c-Abl complex formation. The antiproliferative activity
of IMP, PLP, and the three derivatives was tested against K-562 cells,
showing that the parent compounds had the most pronounced effect on
tumor cells. PLP and IMP were also effective in promoting the c-Abl
nuclear translocation in c-Abl overexpressing cells. Further, these
compounds demonstrated low cytotoxicity on human Hs27 fibroblasts.
In conclusion, our data suggest that 14-3-3σ targeting compounds
represent promising hits for further development of drugs against
c-Abl-dependent cancers.
The ubiquitously
expressed glucocorticoid receptor (GR)
is a nuclear receptor
that controls a broad range of biological processes and is activated
by steroidal glucocorticoids such as hydrocortisone or dexamethasone.
Glucocorticoids are used to treat a wide variety of conditions, from
inflammation to cancer but suffer from a range of side effects that
motivate the search for safer GR modulators. GR is also regulated
outside the steroid-binding site through protein–protein interactions
(PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will
provide insights into noncanonical GR signaling as well as a new level
of control over GR activity. We report the first molecular glues that
selectively stabilize the 14-3-3/GR PPI using the related nuclear
receptor estrogen receptor α (ERα) as a selectivity target
to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect
noncanonical GR signaling and enable the development of novel atypical
GR modulators.
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