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A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

Nature Cell Biology (2024)Cite this article

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Abstract

Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled anchor-away/recruiter system comprises an engineered, 15 kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP–GFP nanobody interaction. Applications of the mitosis-enabled anchor-away/recruiter system include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.

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Fig. 1: PLEKHA5 exhibits increased PM localization during mitosis via PI(4,5)P2 interaction.
Fig. 2: Design and engineering of MARS, based on the H-BP-PH motifs of PLEKHA5.
Fig. 3: The PM localization of PLEKHA5 is regulated by S161 phosphorylation.
Fig. 4: S161 phosphorylation and PLEKHA5 PM localization are modulated by PKC activity.
Fig. 5: MARS recruits PLK1 and PLD to the PM specifically during mitosis.
Fig. 6: MARS-mediated mitotic PM recruitment of Class I PI3K reveals effects of excessive PI(3,4,5)P3 production at the PM during mitosis.

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Data availability

For identification of phosphopeptides, raw mass spectrometry data was processed through the Trans Proteomic Pipeline package (ver. 6.0.0) with spectral data files searched through the Comet search engine over the human UniProt proteome database downloaded on 6 November 2019. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD052237. MARS-related plasmids generated in this study have been deposited in Addgene with the following Addgene IDs: 205232, 205233, 205234, 205235, 205236, 205237, 205238 and 205239. Other data supporting the findings of this study are available within the article and the Supplementary information. Source data are provided with this paper.

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Acknowledgements

We acknowledge support from the National Institutes of Health (NIH) grant R01GM143367 (J.M.B.), NIH grant T32GM138826 (S.H.), NIH grant R35GM141159 (M.B.S.), the Sloan Research Fellowship (J.M.B.) and the DoD DURIP GRANT13369767 and GRANT13710486 (L.B.M.). We thank I. Cheeseman for valuable discussions and for providing the PLK1–GFP cell line, and we thank L. Cantley for sharing the kinase predictions of the PLEKHA5 S161 site. We thank R. Tei, J. Li and N. Vasireddi for technical assistance, the Bretscher lab for the HEK 293TN cell line, the Lammerding lab for the pCDH-CMV-MCS-EF1α-Puro, PAX2 and VSVg plasmids, and access to the IncuCyte system, the Fromme and Emr labs for equipment, and members of the Baskin lab for helpful discussions.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Xiaofu Cao, Shiying Huang, Yuan-Ting Cho, Din-Chi Chiu & Jeremy M. Baskin

  2. Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA

    Xiaofu Cao, Shiying Huang, Mateusz M. Wagner, Yuan-Ting Cho, Din-Chi Chiu, Marcus B. Smolka & Jeremy M. Baskin

  3. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Mateusz M. Wagner & Marcus B. Smolka

  4. Department of Radiology, Weill Cornell Medicine, New York, NY, USA

    Krista M. Wartchow, Artur Lazarian & Laura Beth McIntire

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  1. Xiaofu Cao
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Contributions

Conceptualization: X.C. and J.M.B. Funding acquisition: L.B.M., M.B.S. and J.M.B. Investigation: X.C., S.H., M.M.W., Y.-T.C., D.-C.C., K.M.W. and A.L. Project administration: L.B.M., M.B.S. and J.M.B. Supervision: L.B.M., M.B.S. and J.M.B. Writing—original draft: X.C. and J.M.B. Writing—review and editing: X.C., S.H., M.M.W., Y.-T.C., D.-C.C., L.B.M., M.B.S. and J.M.B.

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Correspondence to Jeremy M. Baskin.

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Extended data

Extended Data Fig. 1 A pool of PLEKHA5 translocates to the PM during mitosis by binding to PI(4,5)P2.

(a) Live-cell confocal microscopy of HeLa cells transiently expressing GFP-tagged PLEKHA5 constructs in interphase and mitosis. Imaging experiment was repeated three times independently with similar results. (b) Representative series of still images from a time-lapse movie in asynchronous, stable H2B-mCherry-expressing (magenta) HeLa cells transfected with PLEKHA5WW-H-BP-PH-GFP (green). Numbers indicate time before/after nuclear envelope breakdown (defined as t = 0 min). Imaging experiment was repeated three times independently with similar results. (c) Schematic representation of proximity biotinylation strategy using Lyn10-TurboID-V5 to biotinylate PM-proximal proteins. (d) Lyn10-TurboID showed expected PM-specific localization and biotinylation activity as demonstrated by staining for V5 (green) and biotin (streptavidin-Alexa Fluor 568, magenta). Imaging experiment was repeated three times independently with similar results. (e) Western blot analysis confirmed that Lyn10-TurboID biotinylates a protein marker of the PM but not markers of other subcellular localizations. The experiment was repeated two times independently with similar results. (f) Clustal Omega alignment of the primary amino acid sequences of the PH domain of PLEKHA4 and PLEKHA5. Highlighted in red are two arginine residues previously established to be required for binding PI(4,5)P2 in PLEKHA4. (g) Confocal microscopy of HeLa cells transfected with the R190A and R244 A mutants of PLEKHA5WW-H-BP-PH-GFP and PLEKHA5H-BP-PH-GFP. Imaging experiment was repeated two times independently with similar results. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 Design and engineering of MARS and MARS-nGFP.

(a) Clustal Omega alignment of primary amino acid sequences around the ‘4A’ region of PLEKHA4 and PLEKHA5. (b) Confocal microscopy of the K157A and R158A mutants of PLEKHA5H-BP-PH-GFP. Imaging experiment was repeated two times independently with similar results. (c, d) Representative series of still images from time-lapse movies in asynchronous HeLa cells stably expressing H2B-tagBFP and transfected with either MARS (c) or 2xMARS-nGFP (d). MARS constructs are colored magenta and H2B-tagBFP is colored green. Numbers indicate time before/after nuclear envelope breakdown (defined as t = 0 min). Each imaging experiment was repeated two times independently with similar results.

Extended Data Fig. 3 Characterization of stoichiometry of 2xMARS-nGFP and effects of MARS systems on cell proliferation rates.

(a) Four stable cell lines were generated via lentiviral transduction that express similar levels of 2xMARS-nGFP and varying levels of GFP, which was under control of either a PGK or CMV promoter (the three CMV-GFP-expressing lines were isolated by FACS, sorting for different levels of GFP expression). Shown are representative Western blots showing GFP (asterisk denotes a longer CMV-GFP product potentially arising from an upstream alternative start codon) and 2xMARS-nGFP (mCherry blot) expression levels in these cell lines alongside blots of purified GFP and mCherry protein standards. (b) Quantification of GFP to 2xMARS-nGFP molar ratios in HeLa cells stably expressing 2xMARS-nGFP and various levels of GFP from (A) (n = 3 biological replicates, one-way ANOVA with Tukey post hoc multiple comparisons test). (c, d) Imaging analysis of GFP recruitment to the PM during mitosis at different stoichiometries of GFP to 2xMARS-nGFP determined by quantitative image analysis. (c) Quantification of the PM to cytosolic fluorescence ratios of GFP as a readout for measuring GFP recruitment levels by 2xMARS-nGFP. For PGK-GFP: n = 8 cells for low GFP expression, n = 4 cells for medium expression, and n = 7 cells for high expression. For CMV-GFP cell lines, n = 10 cells each for low, medium, and high GFP expression level. Cells analyzed came from two independent experiments (one-way ANOVA with Tukey post hoc multiple comparisons test). Note: CMV-GFP cells were sorted by FACS to generate three separate cell lines with low, medium, and high GFP expression, and images were acquired for each cell line. For PGK-GFP cells, the low, medium, and high cells were all from a single cell line and are categorized based on GFP fluorescence intensity during imaging analysis by ImageJ post-acquisition. (d) Representative micrographs for HeLa cells stably expressing 2xMARS-nGFP and varying levels of GFP analyzed in (c). Brightness of the GFP channel was adjusted post-acquisition for better visualization of the weaker fluorescence signals for PGK-GFP and CMV-GFP low cells. (e) Quantification of mean fluorescence intensity in the cytosol during interphase as a means to determine GFP expression levels in PGK-GFP low, medium, high cells and CMV-GFP low cells (n = 40 cells from two independent experiments). Compared to CMV-GFP low cells, PGK-GFP low cells exhibited an average of 7.6x lower expression, PGK-GFP medium cells exhibited ~2.2x lower expression, and PGK-GFP-high cells exhibited ~1.1x higher expression. Converting to GFP:2xMARS-nGFP molar ratio in these cells, the estimated ratios are 1.8 (low), 6.2 (medium), and 15.1 (high). (f, g) Ectopic expression of MARS and 2xMARS-nGFP did not negatively impact rates of cell division and proliferation. (f) Representative growth curves for wild-type (WT) HeLa cells and those stably expressing MARS and 2xMARS-nGFP (n = 2 technical replicates for each curve). Raw data (faded-color curves with error bar) acquired by IncuCyte Live-Cell Analysis System were fitted using GraphPad Prism (Exponential growth curve model). Fitted curves are shown as solid lines. (g) Doubling time of the three cell lines calculated from the fitted growth curves (n = 3 biological replicates, one-way ANOVA with Tukey post hoc multiple comparisons test). Source numerical data and unprocessed blots are available in Source data.

Source data

Extended Data Fig. 4 Mutational analysis reveals no contributions of phosphorylation of residues other than S161 to the cell cycle-dependent localization of PLEKHA5.

Live-cell confocal microscopy of phosphodeficient (Ser/Thr to Ala and Tyr to Phe) and phosphomimetic (Ser/Thr to Asp and Tyr to Glu) mutants of GFP-tagged PLEKHA5 full-length protein (a) or WW-H-BP-PH fragment (b). Imaging experiment was repeated two times independently with similar results.

Extended Data Fig. 5 S161 phosphorylation status influences the PM localization of EGFP-PLEKHA5FL and PLEKHA5WW-H-BP-PH-EGFP.

(a, b) Live-cell confocal microscopy of the subcellular localizations of the WT, S161A, and S161D variants of full-length PLEKHA5FL (a) and the WW-H-BP-PH motifs (b). (c, d) Quantification of PM localization for GFP-PLEKHA5FL (c) and PLEKHA5WW-H-BP-PH-GFP (d) using colocalization analysis. Pearson coefficients were computed from images acquired in HeLa cells transiently expressing either of the PLEKHA5 constructs with a transfectable PM marker, Lyn10-LOV-mCherry. For GFP-PLEKHA5FL in interphase: n = 37 cells for WT, n = 35 cells for S161A, and n = 30 cells for S161D; in mitosis: n = 12 cells for WT and S161D and n = 11 cells for S161A. For PLEKHA5WW-H-BP-PH-GFP in interphase, n = 26 cells for WT, n = 29 cells for S161A, and n = 25 cells for S161D; in mitosis: n = 13 cells for WT and S161D and n = 16 cells for S161A. Cells analyzed came from three independent experiments (one-way ANOVA with Tukey post hoc multiple comparisons test). (e, f) Quantification of PM association for transfected GFP-PLEKHA5FL (e) and PLEKHA5WW-H-BP-PH-GFP (f) using PM-targeted proximity biotinylation. Representative western blots are shown at top with quantification of GFP signals in the streptavidin pulldowns at bottom (n = 4 biological replicates, one-way ANOVA with Tukey post hoc multiple comparisons test). Source numerical data and unprocessed blots are available in Source data.

Source data

Extended Data Fig. 6 Requirement for PI(4,5)P2 binding in S161-mediated PM localization in additional PLEKHA5 constructs and conservation of S161-mediated PM localization in PLEKHA4.

(a, b) Live-cell confocal microscopy of HeLa cells transiently expressing combinational mutants of S161A with either R190A or R244A in PLEKHA5FL-GFP (a) and PLEKHA5H-BP-PH-GFP (b). Imaging experiment was repeated two times independently with similar results. (c) Confocal microscopy of HeLa cells transfected with either the WT, A46S, or A46D forms of full-length PLEKHA4. Imaging experiment was repeated two times independently with similar results. (d) Clustal Omega alignment of amino acid sequences around A46 in PLEKHA4 and S161 in PLEKHA5.

Extended Data Fig. 7 PKC and CK1 kinase activities contribute to S161 phosphorylation.

(a, b) PLEKHA5WW-H-BP-PH-GFP displayed reduced PM localization upon activation of PKC by 100 nM PMA (a) or 200 nM Bryostatin 1 (b). Representative micrographs before and after agonist treatment for each condition are shown at left, with quantifications of the ratios of PM to cytosolic fluorescence (excluding microtubule signal) before and after treatment plotted on the right (n = 12 cells from three independent experiments, one-way ANOVA with Sidak post hoc multiple comparisons test). (c) Top 10 kinase candidates for phosphorylating S161 (highlighted in red) if S159 serves as the priming phosphorylation site. (d) Inhibition of CK1 activity by 5 µM PF670462 or 100 µM D4476 decreased S161 phosphorylation. Shown are representative western blots and quantification for Phos-tag and regular SDS-PAGE analysis of PLEKHA5H-BP-PH-GFP (n = 3 biological replicates, one-way ANOVA with Tukey post hoc multiple comparisons test). (e) Phos-tag SDS-PAGE analysis of S161 phosphorylation upon treatment of inhibitors of the PPP family of protein Ser/Thr phosphatases (Tautomycetin, Fostriecin, and Okadaic acid, each at 1 µM), probing with GFP and phospho-Thr antibodies. The experiment was repeated two times independently with similar results. (f) RT-qPCR analysis of knockdown efficiency for siRNAs targeting CK1 isoforms (CSNK1A1, CSNK1D, CSNK1E), PKC isoforms (PRKCA, PRKCD), and PPP catalytic subunits (PPP1CA, PPP1CB, PPP2CA). (g) Knockdown of CK1 (triple knockdown of CK1a, CK1d, and CK1e) and PKC (double knockdown of PKCa and PKCd) decreased S161 phosphorylation, whereas depletion of PP1 and PP2A (triple knockdown of two PP1catalytic subunits and one PP2A catalytic subunit) increased S161 phosphorylation. Shown are representative Western blots and quantification for Phos-tag and regular SDS-PAGE analysis of PLEKHA5H-BP-PH-GFP (n = 3 biological replicates, unpaired, two-tailed Student’s t-test). Source numerical data and unprocessed blots are available in Source data.

Source data

Extended Data Fig. 8 Evaluation of PM localization of additional MARS constructs for PLD and PI3K and time-lapse imaging of MARS and MARS-p85iSH2 during mitosis.

(a) Live-cell confocal microscopy of HeLa cells transfected with the single MARS fusion to WT or catalytic dead (H170A) forms of PLDPMF revealing only partial recruitment to the PM during mitosis. (b) Representative confocal micrographs of HeLa cells transfected with 2xMARS-nGFP (magenta) and H170A mutant of GFP-PLDPMF (green) showing effective PM recruitment only during mitosis. (c) Live-cell confocal microscopy of HeLa cells transiently expressing the 2xMARS-p85iSH2 construct, revealing undesired partial PM localization during interphase. (d, e) Representative still images from time-lapse movies in asynchronous HeLa cells stably expressing H2B-tagBFP and transfected with either MARS (d) or MARS-p85iSH2 (e), and the PI(3,4,5)P3 marker GFP-AktPH. Images display merged channels of H2B-tagBFP (cyan) and MARS constructs (magenta). Numbers indicate time before/after nuclear envelope breakdown (defined as t = 0 min). All imaging experiments were repeated two times independently with similar results.

Supplementary information

Supplementary Information

Legends for Supplementary Table 1 and Supplementary Videos 1–8. Supplementary Figs. 1–4 and representative gating strategies for flow cytometry experiments.

Reporting Summary

Supplementary Table 1

siRNA duplex sequences and RT–qPCR primer sequences.

Supplementary Video 1

(Related to Extended Data Fig. 1b) Time-lapse confocal microscopy of asynchronous HeLa cells stably expressing H2B-mCherry and transiently transfected with PLEKHA5WW-H-BP-PH–GFP, during mitosis, with cells incubated at 37 °C and images acquired every 1 min. Left: H2B-mCherry. Middle: PLEKHA5WW-H-BP-PH–GFP. Right: merge (mCherry, magenta; GFP, green).

Supplementary Video 2

(Related to Extended Data Fig. 2c) Time-lapse confocal microscopy of asynchronous HeLa cells stably expressing H2B-tagBFP and transiently transfected with MARS, during mitosis, with cells incubated at 37 °C and images acquired every 2 min. Left: H2B-tagBFP. Middle: MARS. Right: merge (tagBFP, green; MARS, magenta).

Supplementary Video 3

(Related to Extended Data Fig. 2d) Time-lapse confocal microscopy of asynchronous HeLa cells stably expressing H2B-tagBFP and transiently transfected with 2xMARS–nGFP, during mitosis, with cells incubated at 37 °C and images acquired every 2 min. Left: H2B-tagBFP. Middle: MARS. Right: merge (tagBFP, green; 2xMARS–nGFP, magenta).

Supplementary Video 4

(Related to Extended Data Fig. 3e) Time-lapse video acquired by the IncuCyte Live-Cell Analysis System of WT HeLa cells incubated at 37 °C with 5% CO2. Images were acquired every 15 min.

Supplementary Video 5

(Related to Extended Data Fig. 3e) Time-lapse video acquired by the IncuCyte Live-Cell Analysis System of HeLa cells stably expressing MARS, with cells incubated at 37 °C with 5% CO2 and images acquired every 15 min.

Supplementary Video 6

(Related to Extended Data Fig. 3e) Time-lapse video acquired by the IncuCyte Live-Cell Analysis System of HeLa cells stably expressing 2xMARS–nGFP, with cells incubated at 37 °C with 5% CO2 and images acquired every 15 min.

Supplementary Video 7

(Related to Fig. 6e and Extended Data Fig. 8d) Time-lapse confocal microscopy of asynchronous HeLa cells stably expressing H2B-tagBFP and transiently transfected with MARS and the PI(3,4,5)P3 marker GFP-AktPH, during mitosis, with cells incubated at 37 °C and images acquired every 4 min. The left video displays merged channels of H2B-tagBFP (cyan) and MARS (magenta), and the right video displays merged channels of H2B-tagBFP (cyan) and GFP-AktPH (yellow).

Supplementary Video 8

(Related to Fig. 6f and Extended Data Fig. 8e) Time-lapse confocal microscopy of asynchronous HeLa cells stably expressing H2B-tagBFP and transiently transfected with MARS–p85iSH2 and the PI(3,4,5)P3 marker GFP-AktPH, during mitosis, with cells incubated at 37 °C and images acquired every 4 min. The left video displays merged channels of H2B-tagBFP (cyan) and MARS–p85iSH2 (magenta), and the right video displays merged channels of H2B-tagBFP (cyan) and GFP-AktPH (yellow).

Source data

Source Data Fig. 1

Statistical source data.

Source Data Figs. 1, 3 and 4 and Extended Data Figs. 1, 3, 5 and 7.

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 3/Table 3

Statistical source data.

Source Data Extended Data Fig. 5/Table 5

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Source Data Extended Data Fig. 7/Table 7

Statistical source data.

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Cao, X., Huang, S., Wagner, M.M. et al. A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01495-8

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