Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 24;115(30):E7053-E7062.
doi: 10.1073/pnas.1803909115. Epub 2018 Jul 11.

Affinity Switching of the LEDGF/p75 IBD Interactome Is Governed by Kinase-Dependent Phosphorylation

Affiliations
Free PMC article

Affinity Switching of the LEDGF/p75 IBD Interactome Is Governed by Kinase-Dependent Phosphorylation

Subhalakshmi Sharma et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Keywords: LEDGF/p75; disordered proteins; leukemia; phosphorylation; protein–protein interactions.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LEDGF/p75 IBD binding partners interact in a structurally conserved manner. (A) Multiple sequence alignment illustrating the conserved LEDGF/p75 IBD binding motif (IBM). Helix-1, the acidic linker, and the FxGF region are indicated below the alignment. (B) Detail of the POGZ IBM–IBD interface from the obtained solution structure. Conserved side chains within POGZ supporting the interaction with IBD are presented as sticks and labeled. The linker region stabilizes the interaction by nonspecific electrostatic contacts with the positively charged patch on the IBD surface. (C) Solution structures of the IBD in complex with the binding motifs from POGZ, JPO2 motif 1 and 2 (M1, M2), IWS1, and MLL1 determined by NMR spectroscopy. (D) Rmsd values comparing structural similarity of each pairwise combination of IBD-bound IBM. (E) The related ARM subdomain of Iws1 from different species interacting with Spt6 (PDB ID codes 3oak and 2xpo, refs. and 35) highlights binding mode similarity to LEDGF/p75 IBD–IBM interactions as exemplified by JPO2 M1.
Fig. 2.
Fig. 2.
Helix-1 in the IBM is essential to maintain interaction with LEDGF/p75 IBD. (A) Diagrams of MLL1, IWS1, and JPO2 constructs and mutants used in B and C. (B) The WT and/or mutant recombinant MLL1, IWS1, and JPO2 protein constructs were titrated against FLAG-tagged LEDGF/p75 in an AlphaScreen assay (counts per second). Error bars represent SDs calculated from independent measurements (n = 4). (C) HEK 293T cells were transfected with FLAG-tagged WT JPO2 and/or the IF-IF mutant. After immunoprecipitation with anti-FLAG antibody the presence of FLAG-tagged proteins and endogenous LEDGF/p75 was confirmed by Western blot.
Fig. 3.
Fig. 3.
Phosphorylation increases the affinity of IBMs for the LEDGF/p75 IBD. (A) Multiple sequence alignment of the LEDGF/p75 IBD binding motif (IBM) highlighting residues modified in BD and F in red. (B) The abundance of phosphorylation in the JPO2, POGZ, MLL1, and IWS1 IBMs as measured by mass spectroscopy. Peptides carrying at least one phospho-group in the acidic linker region and unmodified peptides were quantified. (C) The dissociation constants of WT IBMs compared with phosphorylated or phosphomimetic versions of the peptides. Kd values were obtained by NMR titration. Error bars represent the error of the fit for the 10 most perturbed residues. WT or phosphomimetic mutants of POGZ (D), MLL1 (E), and JPO2 (F) were titrated against FLAG-tagged LEDGF/p75 in AlphaScreen. Error bars represent SDs calculated from independent measurements (n = 4). (G) Densitometry of Western blot bands in H and I. Data are plotted as the ratio of LEDGF/p75 over FLAG-tagged protein, normalized by WT LEDGF/p75. (H and I) HEK 293T cells were transfected with FLAG-tagged WT JPO2 or POGZ and the proteins were immunoprecipitated in the absence (−) or presence (+) of phosphatase inhibitors with anti-FLAG antibody. For FLAG-POGZ, a mutant with modified Ser residues in the IBM region (PogZ S1392A, S1396A) was used as an additional control. The presence of FLAG-tagged proteins and endogenous LEDGF/p75 was confirmed using specific antibodies by Western blot.
Fig. 4.
Fig. 4.
Validation of MED1 as an LEDGF/p75 IBD binding partner. (A) Diagrams of MED1 constructs and mutants used in B. (B) WT and/or mutated recombinant GST-MED1 was titrated against FLAG-tagged LEDGF/p75 in AlphaScreen. Error bars represent SDs calculated from independent measurements (n = 4). (C) Comparison of the 2D 15N/1H HSQC NMR spectra of the IBD in the absence (black) or presence (orange) of MED1847–930. Spectra were obtained from the 15N-labeled recombinant IBD and an unlabeled recombinant MED1 fragment. The residues with the strongest shifts are annotated. (D) IBD amino acid residues that are significantly perturbed upon addition of MED1847–930 (as determined by NMR spectroscopy) are highlighted in orange on the surface of the IBD structure. (E) Representation of the minimal CSP in backbone amide signals of the IBD upon addition of MED1847–930.
Fig. 5.
Fig. 5.
ASK has two IBMs whose affinities for the LEDGF/p75 IBD are differentially regulated by phosphorylation. (A) Diagrams of WT and modified constructs of ASK motif 1 and 2 (M1, M2) used in BE. (B) The WT and/or mutated recombinant ASK protein constructs were titrated against FLAG-tagged LEDGF/p75 in AlphaScreen. Error bars represent SDs calculated from independent measurements (n = 4). (C and D) Comparison of the NMR 2D 15N/1H HSQC spectra of the 15N-labeled IBD in the absence and presence of different ASK synthetic peptides (see also SI Appendix, Fig. S8). (E) IBD amino acid residues that are significantly perturbed upon addition of ASK M1 and phosphorylated ASK M2 (as determined by NMR spectroscopy) are highlighted in red on the surface of the IBD structure. (F) Representation of the minimal CSP in backbone amide signals of the IBD upon addition of ASK-derived peptides.
Fig. 6.
Fig. 6.
IBM phosphorylation by CK2 increases the affinity of POGZ and MLL1 for the LEDGF/p75 IBD. (A) Diagrams of POGZ and MLL1 constructs and mutants used in B and C. (B and C) WT or mutants of POGZ (B) and MLL1 (C) were titrated against FLAG-tagged LEDGF/p75 in AlphaScreen after preincubation with or without CK2. Error bars represent SDs calculated from independent measurements (n = 4). (D) HEK 293T cells were transfected with FLAG-tagged POGZ. The proteins were immunoprecipitated with anti-FLAG antibody in the absence (−) or presence (+) of selective CK2 inhibitor (TBB). The presence of FLAG-tagged POGZ and endogenous LEDGF/p75 was confirmed using Western blot. (E) Densitometry of Western blot bands. Data are plotted as the ratio of LEDGF/p75 over FLAG-POGZ, normalized by DMSO control (mean ± SD from n = 3 independent replicates). P values obtained from Student’s t test; ***P < 0.0001.
Fig. 7.
Fig. 7.
Helix-1 and phosphorylation of flexible linker in MLL1 IBM are both crucial for MLL1-fusion-driven transformation. (A) Diagrams of MLL1 constructs and mutants used in B and C. (B and C) The colony-forming capacity of murine stem cells transformed with an MLL1-ENL fusion was compared with those transformed with MLL1-ENL mutants in helix-1 (B) or in the flexible linker and FxGF motif (C). CFU of lineage-depleted murine hematopoietic stem cells transduced with vectors carrying the WT or mutated MLL1-ENL fusions were determined in three rounds. Errors bars indicate SDs determined from independent replicates (n = 3). Statistical differences were determined using Student’s t test; *P < 0.05; ns, nonsignificant compared with WT. (D) Summary and conceptual model of IBM affinity switching.

Similar articles

See all similar articles

Cited by 5 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback