The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

Abstract

The su(var)3‐9, enhancer of zeste, trithorax (SET)/inhibitor 2 of protein phosphatase 2A (PP2A) oncoprotein binds and inhibits PP2A, composed of various isoforms of scaffolding, regulatory, and catalytic subunits. Targeting SET with a sphingolipid analog drug fingolimod (FTY720) or ceramide leads to the reactivation of tumor suppressor PP2A. However, molecular details of the SET‐FTY720 or SET‐ceramide, and mechanism of FTY720‐dependent PP2A activation, remain unknown. Here, we report the first in solution examination of the SET‐FTY720 or SET‐ceramide complexes by NMR spectroscopy. FTY720‐ceramide binding resulted in chemical shifts of residues residing at the N terminus of SET, preventing its dimerization or oligomerization. This then released SET from PP2ACα, resulting in PP2A activation, while monomeric SET remained associated with the B56γ. Our data also suggest that the PP2A holoenzyme, composed of PP2A‐Aβ, PP2A‐B56γ, and PP2ACα subunits, is selectively activated in response to the formation of the SET‐FTY720 complex in A549 cells. Various PP2A‐associated downstream effector proteins in the presence or absence of FTY720 were then identified by stable isotope labeling with amino cells in cell culture, including tumor suppressor nonmuscle myosin IIA. Attenuation of FTY720‐SET association by point mutations of residues that are involved in FTY720 binding or dephosphorylation of SET at Serine 171, enhanced SET oligomerization and the formation of the SET‐PP2A inhibitory complex, leading to resistance to FTY720‐dependent PP2A activation.—De Palma, R. M., Parnham, S. R., Li, Y., Oaks, J. J., Peterson, Y. K., Szulc, Z. M., Roth, B. M., Xing, Y., Ogretmen, B. The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction. FASEB J. 33, 7647–7666 (2019). http://www.fasebj.org

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“…Furthermore, our simulation results showed that the ceramide molecule adopts an "extended" conformation inside the SET protein, in which the sphingosine and lipid chains are placed in opposite directions. This finding was further confirmed by the NMR model suggested previously by De Palma et al [41]. The docking results of compound 1 revealed that the ligand exhibited a similar extended orientation and comparable binding interactions within the pocket (S = −6.6821 kcal/mol).…”

“…Additionally, along the N-terminal region, the 1,4-dihydroxy array of the dihydrosphingosine chain donates two H-bonds to Glu-111 (2.843 and 2.849 Å, respectively), while the 3-hydroxy group accepts an H-bond from Gln-65 with 2.138 Å ( Figure 6A). Previous data had indicated that the amino acid residues 36-124 of the N-terminal region are involved in the inhibition of PP2A as well as the binding with ceramide [41]. On the other hand, compound 2 was found to exhibit a different compact conformation within the active site, where the dihydrosphingosine backbone runs perpendicular to both the fatty acyl chain and the sugar moiety.…”

“…Modeling and docking simulations of the newly isolated compounds (1 and 2) were performed using the Molecular Operating Environment (MOE) software [39] and the crystal structure of Su(var)3-9, enhancer of Zeste, Trithorax (SET)/inhibitor 2 of protein phosphatase 2A (I2PP2A) oncoprotein; (PDB: 2E50) [40]. D-erythro(e)-C 18 ceramide was chosen as a reference compound in the simulation studies due to its higher affinity for binding to SET protein compared to other endogenous ceramides or sphingosine [41]. PP2A has been reported to exhibit a tumor suppressive function by inducing apoptosis or programmed cell death in various cancerous cells [42].…”

“…Sphingolipid ceramide has long been described to activate PP2A through a direct interaction with the PP2AC catalytic domain [43], but its mechanistic details have so far remained unknown. Alternatively, another mechanism of ceramide-induced activation of PP2A has been recently found to proceed through the direct binding of ceramide with SET oncoprotein, which functions as an inhibitor of tumor suppressor PP2A [41]. Because the crystal structure of the ceramide-SET complex was not available in the protein data bank, induced fit docking was performed using the crystal structure of the SET protein and C 18 ceramide as a flexed ligand.…”

The Role of MYC and PP2A in the Initiation and Progression of Myeloid Leukemias

“…In AML, SET is highly dependent on MYC-transcriptional activity and recruitment of RUNX1 and GATA2 on its promoter [102]. By impairing SET binding to PP2A, with molecules like FTY720 [82,105], CM-1231 [106], or OP449 [107,108] it is possible to re-establish PP2A activity, inhibiting tumor growth [91,109] and overcoming therapeutic resistance in preclinical models [92,108]. SET is known to interact with SETBP1 (SET-binding protein 1), which protects SET from protease cleavage.…”