Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

Abstract

Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

Keywords: CRISPR screening; CRISPRi; GSK3; click chemistry; phospholipase D.

Fig. 1.

Combining pooled CRISPRi screening with bioorthogonal labeling to visualize phospholipase D signaling. (A) Scheme for pooled CRISPR screening based on bioorthogonal labeling to uncover regulators of the pathway of interest targeted by the bioorthogonal labeling step. (B) PLD-mediated PA synthesis and IMPACT method for detecting PLD activity. Top arrow: PLD signaling involves hydrolysis of phosphatidylcholine to generate the lipid second messenger PA. Bottom arrow: IMPACT as a means to generate fluorescent reporters of PLD signaling. IMPACT takes advantage of PLD-catalyzed transphosphatidylation with 3-azidopropanol followed by click chemistry tagging via strain-promoted azide-alkyne cycloaddition with a fluorescent cyclooctyne reagent (BCN-BODIPY) to generate fluorescent phosphatidyl alcohol reporters to enable FACS-based enrichment of cells based on their PLD activity.

Fig. 2.

Combining IMPACT with genome-wide CRISPRi screening to identify regulators of protein kinase C–PLD signaling. (A) Experimental overview. A population of ∼125 million K562-dCas9-KRAB (K562i) cells was transduced at an MOI of ∼0.4 with a lentiviral sgRNA library of sgRNAs targeting the entire human genome. PKC-mediated PLD signaling was then stimulated by the addition of PMA, and IMPACT labeling was performed to fluorescently label cells according to PLD signaling activity. The top and bottom quartiles of the labeled population were collected via FACS, and enriched sgRNAs in those populations were identified by Illumina next-generation sequencing. (B) Results from the screen. Volcano plots depict the three pairwise comparisons of sgRNA levels in high IMPACT, low IMPACT, and unsorted cell populations. X-axes show ratio of sgRNA abundance in the indicated populations, y-axes show the Mann–Whitney U test P value. (Insets) Boxed region, enlarged for clarity. Green data points: genes predicted to be “activators” of PLD; red data points: genes predicted to be “inhibitors” of PLD. Shown below is a combined listing of all putative activators and inhibitors from the screen. See SI Appendix, Table S1 for separate lists of hits from each pairwise comparison (i.e., from each volcano plot). (C) Validation of several putative regulators of PLD signaling. K562i cells expressing CRISPRi sgRNA targeting the indicated gene (shown in boxes in volcano plot) were labeled by IMPACT with PMA stimulation, and flow cytometry analysis was performed. Plotted are the mean fluorescence intensities of the labeled populations, with background fluorescence subtracted and normalized to control sgRNA (gray). n = 8 to 21, ANOVA (Tukey) *P < 0.05; **P < 0.01, ****P < 0.001.

Fig. 3.

GSK3 regulates PA-dependent signaling via the expression of PLD and PKC. (A and B) MDA-MB-231 cells were treated with the GSK3 inhibitor LY2090314 (20 nM, blue) or vehicle (dimethyl sulfoxide [DMSO], black) for the indicated length of time and then subjected to IMPACT labeling by the addition of 3-azidopropanol (1 mM) and PMA (100 nM), rinsing, and strain-promoted azide-alkyne cycloaddition tagging with BCN-BODIPY. Lipid extracts were generated, and the IMPACT-derived fluorescent lipids were detected and quantified using fluorescence-coupled HPLC. (C) MDA-MB-231 cells were treated with LY2090314 (20 nM) or DMSO vehicle for 24 h. Protein lysates were collected, analyzed, and quantified via Western blot. (D) MDA-MB-231 cells were treated with LY2090314 (20 nM) or DMSO vehicle for 24 h. Cellular messenger RNA was collected, converted to complementary DNA via reverse transcription, and quantified via qPCR. ΔΔCp values were calculated by comparison to a tubulin control. Plotted are relative messenger RNA levels (2^-ΔΔCp) for each transcript of interest in cells treated with LY2090314 as compared to those treated with DMSO vehicle. (E) Model for how GSK3 activity promotes the transcription of PKCα and PLD1/2, causing their levels to increase and priming cells to have elevated agonist-induced PKC–PLD-mediated PA production and signaling. To attenuate PA synthesis and maintain homeostasis, an effect of PKC activation is its phosphorylation and inactivation of GSK3, thus reducing the transcription of PKC and PLD to reset the system. Statistical analysis: (A) n = 14, ****P = 1.5 × 10⁻¹⁴, Student’s t test. (B and C) n = 6 (B) and n = 3 (C), ANOVA with post hoc Tukey test; *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. (D) n = 6. Error bars: 1σ shown around the mean in A–C and 95% CI in D.