1. Volume 63, Issue 4, Pages 686-695 (August 2016)
A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase 
Emil Peter Thrane Hertz, Thomas Kruse, Norman E. Davey, Blanca López-Méndez, Jón Otti Sigurðsson, Guillermo Montoya, Jesper V. Olsen, Jakob Nilsson 
Molecular Cell 
Volume 63, Issue 4, Pages (August 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 63, 686-695DOI: (10.1016/j.molcel.2016.06.024)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
The PP2A-B56 Holoenzyme Binds to Short Linear LxxIxE Motifs Present throughout Eukaryotes
(A) Volcano plot representing mass spectrometry-identified proteins co-purifying with YFP-B56α (blue dots) versus YFP-B55α (red dots) from asynchronously growing HeLa cell lines stably expressing YFP-B56α or YFP-B55α.
(B) Domain organization and alignment of amino acid residues (boxed) required for KIF4A binding to B56.
(C) Alanine scanning mutagenesis through the C terminus of KIF4A. The indicated Myc-KIF4A (amino acids 1001–1232) derivatives were transfected into HeLa cells stably expressing YFP-B56α. YFP-B56 was purified (IP) and KIF4A binding determined. PP2A-C, catalytic subunit; input for IP, In.
(D) Alignment of LxxIxE motifs validated here or elsewhere (BubR1, Repo-Man) with binding affinity of individual LxxIxE motifs as indicated. ND, not determined.
(E) Anatomy of the LxxIxE motif. The core determinants in P1, P4, and P6 are listed and ordered according to the affinity contribution they provide to B56 binding. Phosphorylation of residues, or the presence of acidic residues, in the indicated positions in the motif increases the affinity to B56.
(F) Overview of the workflow of the in silico approach to identify LxxIxE motifs.
See also Figures S1–S5 and Tables S1 and S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 2
Recognition of LxxIxE Motifs by a Conserved Binding Pocket on B56 Regulatory Subunits
(A) Peptide competition assay using a wild-type GEF-H1 peptide (WT) or a mutated variant (mutant) unable to bind B56 used at 18 or 180 μM final concentration. Lysates from mitotic shake-off cells were prepared from control (Ctrl) HeLa cells or cells stably expressing YFP-B56α. Competing GEF-H1 peptides were added to cell lysates 30 min prior to YFP-B56 purification and binding of indicated proteins determined.
(B) Amino acid conservation of B56 orthologs. • indicates amino acid residues critical for binding of B56 to the LxxIxE motif.
(C) IP of YFP-B56α from cells stably expressing the indicated B56α variants and subsequent immunoblotting of indicated proteins. PP2A-C, PP2A catalytic subunit; PP2A-A, PP2A scaffold subunit.
(D) The amino acid conservation map of B56 is displayed on the surface model of B56γ (PDB: 3FGA) (Landau et al., 2005). The electrostatic potential map in the middle panel indicates the basic character of the highly conserved region. A detailed view of the consensus motif LxxIxE of the KIF4A C1224L peptide docked in the pocket of the B56 (B56α nomenclature) model is in the far right panel.
See also Figure S5D.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 3 LxxIxE Motifs Regulate Phosphorylation Status of RacGAP1
(A) Domain organization of RacGAP1 and reported phosphorylated serines by Plk1 (black triangles).
(B) Depletion efficiency of endogenous RacGAP1 (black arrow) using RNAi. Expression of doxycycline-inducible RacGAP1-YFP variants (white arrow) in stable HeLa Flp-In T-REx cell lines. Doxycycline was added simultaneously with RacGAP1 siRNA and cell lysates prepared from nocodazole-treated cell 48 hr post-transfection. GAPDH was used as loading control.
(C) Live-cell imaging of cells expressing wild-type (WT) RacGAP1-YFP or RacGAP1-2A-YFP (L143A-I146A) as they go through mitosis. Circumference of cells in the right-most panels are marked by dashed lines to highlight the occurrence of mono-nuclear or bi-nuclear cells.
(D) Immunolocalization images of RacGAP1-YFP and RacGAP1-2A-YFP on the central spindle in anaphase cells depleted of endogenous RacGAP1 and stained with serine157 or serine170 phospho-specific antibodies as indicated. Scale bar, 1 μM.
(E) Quantification of (D). Each data point represents quantification from a single cell.
(F) The indicated RacGAP1-YFP constructs were purified using GFP-Trap and analyzed for phosphorylation by immunoblotting. λ-phos, λ-phosphatase treated.
(G) Quantification of (F).
(H) Quantification of live-cell imaging experiments, monitoring the generation of bi-nucleated cells in RacGAP1 RNAi rescue experiments. Data are represented as mean ± 99% confidence interval from n > 214 pooled for each condition. ∗∗∗∗p < using a two-tailed unpaired t test.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 4 Regulation of FOXO3 Localization by Engineered LxxIxE Motifs
(A) Binding affinities of FOXO3 peptides with engineered LxxIxE motifs. NB, not binding.
(B) A stable HeLa cell line expressing YFP-B56α was transfected with the indicated myc-tagged FOXO3 constructs. Cell lysate from asynchronous cells was incubated with GFP-Trap resin. The amount of co-purifying myc-FOXO3 was determined by immunoblotting using anti-Myc antibody.
(C) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells.
(D) Quantification of (C). Each data point represents quantification from a single cell. Mann-Whitney test significance values; ∗∗p < 0.01, ∗∗∗∗p <
(E) Translocation kinetics after Akt inhibition measured by live-cell imaging. Only cells showing comparative initial YFP-FOXO3 nucleo-cytoplasmic ratios were selected for analysis. Images were acquired every 3 min and the intensity of nuclear and cytoplasmic YFP measured.
(F) Quantification of (E). A minimum of eight cells were measured for each time point. Data are represented as mean ± SEM.
(G) The indicated YFP-FOXO3 variants were purified from asynchronous cells and analyzed for phosphorylated Akt (RxxpS/T) or phosphorylated sites (LxRxxpS/T). λ-phos., λ-phosphatase treated.
(H) Quantification of (G).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2016 Elsevier Inc. Terms and Conditions