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Modulation of PHLPP activity: screening for specific inhibitors
Author(s) -
Sierecki Emma,
Newton Alexandra C
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb289
Subject(s) - protein kinase b , pleckstrin homology domain , chemistry , phosphatase , phosphorylation , biology , biochemistry
PHLPP (PH domain Leucin repeats Protein Phosphatase) has been shown to directly dephosphorylate Akt and PKC, leading to the desactivation of the Akt pathway and degradation of PKC. PHLPP also inhibits the Erk pathway and regulates the levels of tyrosine kinase receptors, in an indirect manner. In order to decipher the role of PHLPP in these different pathways, we need chemical tools to modulate PHLPP activity. However, the phosphatase domain of PHLPP belongs to the PP2C family of phosphatases that are characterized by their requirement of manganese/magnesium for their activity and by their resistance to commonly used phosphatase inhibitors such as okadaic acid or calyculin. To find specific inhibitors and activators, we screened the Diversity Set of the NCI for inhibitors of PHLPP 2. We designed a medium throughput colorimetric assay, using pNPP as a substrate. Compounds were tested at 100 µM and cutoff was set at 30% activity. We then evaluated the specificity of the compounds against the other member of the PHLPP family, PHLPP 1 and against another member of the PP2C family, PP2Cα. We determined the IC 50 for the most promising compounds. Molecules with a IC 50 of ≈1µM were also tested in cells and their effect on PHLPP was accessed by monitoring the phosphorylation state of Akt. This work was supported by the JDFR 3‐2008‐478 and NIH 067946.