Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration

This information is current as Xiaolu Xu, Xinxin Wang, Elizabeth M. Todd, Emily R. of September 25, 2021. Jaeger, Jennifer L. Vella, Olivia L. Mooren, Yunfeng Feng, Jiancheng Hu, John A. Cooper, Sharon Celeste Morley and Yina H. Huang J Immunol 2016; 197:1683-1691; Prepublished online 27

July 2016; Downloaded from doi: 10.4049/jimmunol.1600874 http://www.jimmunol.org/content/197/5/1683

Supplementary http://www.jimmunol.org/content/suppl/2016/07/26/jimmunol.160087 http://www.jimmunol.org/ Material 4.DCSupplemental References This article cites 43 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/197/5/1683.full#ref-list-1

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Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration

Xiaolu Xu,*,1 Xinxin Wang,*,2 Elizabeth M. Todd,† Emily R. Jaeger,*,3 Jennifer L. Vella,‡ Olivia L. Mooren,x,{ Yunfeng Feng,‖ Jiancheng Hu,*,4 John A. Cooper,x,{ Sharon Celeste Morley,*,†,5 and Yina H. Huang‡,‖,5

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20–like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream Downloaded from of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr89. We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr89 to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell http://www.jimmunol.org/ migration. The Journal of Immunology, 2016, 197: 1683–1691.

ffective immune surveillance requires continuous traf- and adhesion (8–10). Mst1 activity is required in T cells for ficking of T cells among the blood, lymphatic system, and chemokine-induced integrin activation and distribution (11, 12), E secondary lymphoid organs (1). T cell trafficking is regu- cellular polarization (13), and thymic egress (14, 15). Despite the lated by chemoattractants, which provide directional cues by bind- importance of Mst1 to T cell migration, downstream effector path- ing to G-protein–coupled receptors and inducing T cell polarization ways are largely unexplored. Mst1 activates the classical hippo

(2, 3). T cell polarization includes formation of a lamellipodium, a pathway components Mob1/Lats, which indirectly promote Rab13- by guest on September 25, 2021 flat fanlike structure supported by a highly branched F-actin net- mediated integrin clustering (11) and DOCK8-facilitated actin po- work, at the leading edge. The signaling intermediaries that translate lymerization (15). However, no direct link between Mst1 and an chemoattractant binding into changes in the F-actin network have actin-binding protein has been described. not yet been fully elucidated. Multiple actin-binding proteins help to create and stabilize One signaling molecule essential for T cell migration is the mam- lamellipodial F-actin, including the actin-binding protein L-plastin malian Ste20–like kinase 1 (Mst1), also known as STK4 in humans (LPL). LPL is an abundantly expressed immune-specific actin- and Hippo in Drosophila. Mst1 mutations cause both a primary bundling protein of the a-actinin family. LPL is enriched in the immunodeficiency disease (4–6) and an autoimmune syndrome in lamellipodium during migration (16–18). Deficiency of LPL impairs humans (7) and mice due to defects in T cell activation, trafficking, T cell polarization, lamellipodial formation, and T cell migration

*Department of Pathology and Immunology, Washington University School of Med- The work was supported by National Institutes of Health (NIH) Grants GM38542 and icine, St. Louis, MO 63110; †Division of Infectious Diseases, Department of GM118171 (to J.A.C.) and AI104732 (to S.C.M.). S.C.M. is supported by Children’s Pediatrics, Washington University School of Medicine, St. Louis, MO 63110; Discovery Institute Grant MD-FR-2010-83, Basil O’Connor Starter Scholar Research ‡Department of Microbiology and Immunology, The Geisel School of Medicine at Award Grant 5-FY13-192, the March of Dimes Foundation, an American Heart Dartmouth, Lebanon, NH 03756; xDepartment of Biochemistry and Molecular Bio- Association Grant-in-Aid, and Child Health Research Center of Excellence in physics, Washington University School of Medicine, St. Louis, MO 63110; {Depart- Developmental Biology at Washington University School of Medicine Grant ment of Cell Biology and Physiology, Washington University School of Medicine, K12-HD076224. Y.H.H. is supported by NIH Grants AI089805 and S10RR024688 St. Louis, MO 63110; and ‖Department of Pathology, The Geisel School of Medicine (to Duane Compton, Dartmouth College). at Dartmouth, Lebanon, NH 03756 Address correspondence and reprint requests to Dr. Sharon Celeste Morley or 1Current address: Yale School of Medicine, New Haven, CT. Dr. Yina H. Huang, Departments of Pediatrics and Pathology and Immunology, Washington University School of Medicine, CB 8208, 660 South Euclid Avenue, 2Current address: Department of Immuno-Oncology, Poseida Therapeutics, Inc., San St. Louis, MO 63110 (S.C.M.) or Departments of Pathology and Microbiology and Diego, CA. Immunology, The Geisel School of Medicine at Dartmouth, HB 7600, Borwell 650E, 3Current address: Saint Louis University School of Medicine, St. Louis, MO. One Medical Center Drive, Lebanon, NH 03756 (Y.H.H.). E-mail addresses: [email protected] (S.C.M.) or [email protected] (Y.H.H.) 4Current address: Division of Cellular and Molecular Research, National Cancer Center Singapore and Cancer & Stem Cell Program, Duke-NUS Medical School, The online version of this article contains supplemental material. National University of Singapore, Singapore. Abbreviations used in this article: CaM, calmodulin; CD4SP, CD4 single-positive; 5S.C.M. and Y.H.H. are cosenior authors. CD8SP, CD8 single-positive; C-Luc, C-terminal luciferase; HSC, hematopoietic stem cell; LPL, L-plastin; Mst1, mammalian Ste20–like kinase 1; N-Luc, N-terminal ORCIDs: 0000-0002-9940-3605 (X.X.); 0000-0002-6190-8114 (J.L.V.); 0000-0001- luciferase; Wt, wild-type. 9150-7795 (Y.F.); 0000-0003-1615-0030 (J.H.); 0000-0002-0933-4571 (J.A.C.); 0000-0001-8424-0121 (S.C.M.); 0000-0002-0125-9351 (Y.H.H.). Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Received for publication May 19, 2016. Accepted for publication June 27, 2016. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600874 1684 LPL IS A DOWNSTREAM EFFECTOR OF Mst1 IN T CELLS

(17, 19). The regulatory elements that control localization and (Stratagene, La Jolla, CA) according to the manufacturer’s manual. For function of LPL have not been fully described. LPL contains an purification of rLPL and Mst1 kinase domain, pGEX constructs of LPL N-terminal regulatory headpiece followed by two C-terminal actin- and Mst1 kinase domain were generated according to the manufacturer’s manual (GE Healthcare, Piscataway, NJ). For the luciferase complementa- binding domains. Potential regulatory elements in the headpiece tion assay, Mst1 kinase domain was fused in front of the N-lobe of luciferase include a serine phosphorylation site (Ser5), two EF calcium-binding and LPL behind the C-lobe of luciferase in two separate constructs gener- domains, and a calmodulin-binding domain (20). Phosphorylation of ously provided by Dr. David Piwnica-Worms (The University of Texas MD Ser5 increases actin-bundling activity (21), whereas calcium binding Anderson Cancer Center, Houston, TX). decreases the interactions of LPL with F-actin (22). Calmodulin Luciferase complementation assay binding supports the localization of LPL to and maintenance of the Luciferase complementation assays were carried out as described (27). immunological synapse (23). However, none of these elements has Briefly, 293T cells were cotransfected with different combinations of Mst1– been shown to definitively regulate localization of LPL to the N-terminal domain of luciferase (N-Luc) and target–C-terminal domain of lamellipodia of migrating T cells. luciferase (C-Luc) fusion constructs with FuGENE 6 (Promega, Madison, In this study, we identify LPL as a direct cytoskeletal target of WI). One day posttransfection, cells were seeded into luciferase plates. Luciferin substrate was added after 12 h and imaged using an IVIS-200 Mst1-mediated phosphorylation. The regulatory headpiece of LPL in vivo imaging system (Caliper Life Sciences, Hopkinton, MA). contains a consensus Mst1 phosphorylation site, and we show that Alignment of LPL sequences Mst1 kinase phosphorylates the Thr89 residue (T89) of LPL. To test the requirement for phosphorylation of T89 during T cell migration, NetPhorest (http://netphorest.info/) (28) was used to search for potential we re-expressed a T89A mutant of LPL in LPL-deficient T cells. MST phosphorylation sites computationally. Alignment of plastins was performed using ClustalW2 and Clustal V (http://www.ebi.ac.uk/Tools/ Downloaded from The mutant T89A LPL did not rescue lamellipodial formation and msa/clustalo/). in vitro T cell migration when expressed in LPL-deficient T cells, whereas expression of wild-type (Wt) LPL did. Finally, expression Purification of recombinant protein of T89A LPL in bone marrow chimeras also failed to rescue the GST-Mst1 kinase domain and LPL protein were expressed in Escherichia phenotype of impaired thymic egress found in LPL2/2 mice. Thus, coli BL21. Briefly, cells were transformed with a pGEX construct and we identify LPL as a direct target of Mst1. Our results reveal a new grown overnight before induction with IPTG for 1–3 h. The cells were suspended in lysis buffer (1% Triton X-100, 1 mM EDTA [pH 8], lysozyme, mechanism by which Mst1 controls higher-order actin structures. protease inhibitors [Roche, Indianapolis, IN], and 50 mM Tris-Base [pH 8]) http://www.jimmunol.org/ and lysed with repeated freeze-and-thaw cycles in liquid nitrogen and a 37˚C water bath. The lysate was sonicated to fragment bacterial DNA. The lysate Materials and Methods was clarified and incubated with glutathione beads (GE Healthcare) for 6 h. Mice Protein was eluted with reduced glutathione (Sigma-Aldrich) overnight. Mst1h/h and LPL-deficient mice have been previously described (12, 24). In vitro kinase assay In most experiments, adult male and female mice were used. Because thymic profiles vary with age and sex, only data from adult male mice are Purified rGST-Mst1 kinase domain and GST-LPL were mixed together in the presence of kinase buffer (25 mM HEPES, 10 mM MgCl2, 0.5 mM displayed in Fig. 1C. Mice were housed in a specific pathogen-free facility 32 under the supervision of the Division of Comparative Medicine at Washington NaVO4, and 0.5 mM DTT, plus 100 mM ATP) with or without [g-[ P]]- University School of Medicine. Animal studies were approved by the ATP. Reactions were terminated after 45 min with SDS-PAGE sample by guest on September 25, 2021 buffer and boiled for 1 min before separation by SDS-PAGE. In cases in Washington University and Dartmouth College Institutional Animal Care 32 and Use Committees. which g-[ P]]-ATP was added, the gel was stained with Coomassie blue and visualized by autoradiography. Abs Immunoblot analysis Abs for immunoblot analysis were as follows: LPL (Santa Cruz Biotechnology, CD4+ T cells were purified from mouse spleen and lymph nodes with Dallas, TX), Mst1 and phospho–Thr-X-Arg (Cell Signaling Technology, Dynabeads Untouched Mouse CD4 kit (Life Technologies, Carlsbad, CA). The Danvers, MA), and b-actin (Sigma-Aldrich, St. Louis, MO). The following purified cells were then rested in 37˚C for 20 min. Cells were then stimulated Abs used for flow cytometric analysis were purchased from BioLegend (San with CCL19 (100 ng–1 mg/ml) and lysed with actin cytoskeleton-preserving Diego, CA) and eBioscience (San Diego, CA): anti-CD45.1–PerCPCy5.5, lysis buffer (0.1% Triton X-100, 2 mM MgCl2, 150 mM KCl, 10 mM HEPES, anti-CD45.2–Alexa 700, anti-CD4–allophycocyanin, anti-CD8–PECy7, anti- and Mini-complete phosphatase and protease inhibitors [Roche]), standard CD24–Pacific Blue, and anti-CD69–PE. lysis buffer (1% Triton X-100, 150 mM NaCl, 40 mM Tris [pH 7.5], and Mini- + complete phosphatase and protease inhibitors), or 13 NuPAGE LDS Sample Immunofluorescence of CD4 lymphocytes buffer (Life Technologies) with reducing agent. Cell lysates were centrifuged Purified CD4+ T cells were nucleofected with a Lonza Nucleofector 2b and the supernatants analyzed by SDS-PAGE and immunoblot. system (Lonza) with a LifeAct-Ruby expression plasmid (Addgene) (25) 1 d In vitro actin bundling assay prior to seeding on ICAM-1–coated chamber slides. T cells were stimulated with chemokine (CCL19 at 100 ng/ml) prior to fixation with 4% parafor- Alexa 488–conjugated rabbit muscle actin mixed 30/70 with unlabeled maldehyde. Images were captured using either a WaveFX-X1 spinning disk actin was polymerized for 20 min in actin polymerization buffer (Cyto- confocal microscope (Quorum Technologies, Guelph, ON, Canada) equipped skeleton, Denver, CO) at a concentration of 3 mM followed by flow ad- with a Hamamatsu ImageEM camera (Hamamatsu Photonics) or an Olym- dition of kinase buffer alone, rLPL (250 nmol) in kinase buffer, or rLPL pus IX81-ZeroDrift 2 inverted microscope (Olympus) equipped with wide- and Mst1 in kinase buffer. Actin filaments and bundles were visualized by field fluorescence light source and shutters, celltirf TIRFM illuminator TIRF microscopy (Olympus Life Science), as described above. (Olympus Life Science) using a 603 1.49 numerical aperture oil objective, and an Andor Zyla 5.5 camera (Andor). Images were initially processed in Transwell assay Fiji (26) for conversion to multichannel TIF files. Purified CD4+ T cells were seeded into top chambers over 5-mmTranswell Images of cells were randomized and scored by independent blinded inserts with 100 ng/ml CCL19 (PeproTech) in the lower chamber. After 1.5 h observers for: 1) enrichment of LPL in the lamellipod; and 2) appearance of at 37˚C, cells were recovered from the lower chamber and counted by high- F-actin in either a clumped or smooth morphology (Fig. 4). throughput enabled flow cytometer LSR II (BD Biosciences). Percentage of migrated cells was determined as a percentage of total input. In some cases, Constructs and cloning the transwell inserts were precoated with BSA or 2 g/ml ICAM-1–Fc (R&D Lentiviral constructs for expression of LPL were generated with pLVX Systems). (Clontech, Mountain View, CA) as the backbone and the LPL coding region Generation of bone marrow chimeras amplified from pMX-LPL plasmid generously provided by Dr. Eric Brown (MTA OR-210216; Genentech) with PCR. Site-directed mutagenesis to Hematopoietic stem cells (HSCs) were purified from LPL2/2 mice using generate mutant LPL constructs was carried out using the Quikchange kit the Miltenyi Biotec anti–c-Kit positive selection kit (Miltenyi Biotec) The Journal of Immunology 1685 according to the manufacturer’s manual, followed by FACS of c-Kit+Sca1+ cells. HSCs were allowed to proliferate in the presence of stem cell factor (50 ng/ml) and thrombopoietin (50 ng/ml) and transduced with lentivirus to express GFP-LPL. Transduced HSCs (.10,000/recipient) were injected i.v. into lethally irradiated CD45.1+ congenic mice. Eight weeks following reconstitution, thymus was harvested from recipients and analyzed by flow cytometry. Statistical analysis All data were analyzed and graphed using Prism (GraphPad Software, La Jolla, CA). Comparisons of two groups were made using nonparametric Mann–Whitney test, and comparisons of more than two groups were made using one- or two-way ANOVA. The p values for binomial data (e.g., Fig. 4C) were determined using Fisher exact test.

Results Mst1h/h and LPL2/2 mice are phenotypically similar The phenotypes of Mst1 kinase deficiency and LPL deficiency in mice are similar, with reduced thymic egress, reduced peripheral T cell numbers, and reduced T cell migration (18, 29). To begin Downloaded from our analysis, we first directly compared Mst1-hypomorphic [Mst1h/h (12)] and LPL2/2 mice in phenotypic assays (Fig. 1A, 1B). Splenic CD4+ and CD8+ T cell numbers (Fig. 1A) and CCL19-dependent CD4 T cell migration (Fig. 1B) were decreased in Mst1h/h and LPL2/2 mice compared with Wt mice. We also confirmed dimin- 2/2 ished lamellipodial formation in T cells from LPL mice (Fig. 1C, http://www.jimmunol.org/ 1D) (17), resembling the phenotype reported in Mst1h/h mice (12). Together, the similarities in the phenotypes suggest that Mst1 and LPL may function in the same regulatory pathway. To evaluate whether Mst1 and LPL function in the same pathway, we tested for a genetic interaction by asking whether combined haploinsufficiency of Mst1 and LPL impaired thymic egress to a greater extent than haploinsufficiency of either alone. Decreased h/h 2/2 thymic egress, observed in both Mst1- and LPL-deficient mice, is FIGURE 1. The phenotypes of Mst1 and LPL mice are similar. + + 2/2 A by guest on September 25, 2021 characterized by increased proportions of mature CD4 single- ( ) Total numbers of CD4 and CD8 T cells in spleens of Wt, LPL , and Mst1h/h mice. (B) Migration efficiency of CD4+ T cells from Wt, positive (CD4SP) and CD8 single-positive (CD8SP) thymocytes, 2/2 h/h neg neg LPL , and Mst1 mice. Cells were incubated in the upper well of 5-mm which are identified as CD69 and CD24 (14, 15, 19). Analysis Transwell membrane chambers; the bottom well contained 100 ng/ml of mice heterozygous for the Mst1 hypomorphic allele and for LPL CCL19. Data are representative of at least two independent experiments. +/h +/2 deficiency (Mst1 LPL mice) revealed significant accumulation (C) Impaired lamellipodial formation in T cells from LPL2/2 mice. Arrow +/h of mature CD4SP and CD8SP thymocytes compared with Wt, Mst indicates flat lamellipodium at leading edge of Wt cell. Representative 2 and LPL+/ mice (Fig. 1E). This genetic interaction suggests that images. (D) Quantification of percentages of T cells from WT and LPL2/2 Mst1 and LPL function in the same pathway. mice that generate lamellipodia, from three independent experiments. p value determined using Fisher exact test. (E) Increased proportions of Mst1 and LPL can interact in cells CD4SP and CD8SP thymocytes from Mst1+/h LPL+/2 mice exhibit a neg neg To evaluate whether the Mst1 and LPL proteins are able to interact mature (CD69 CD24 ) phenotype, indicating reduced thymic egress. in cells, we performed luciferase complementation assays (27). Full- Each symbol represents data from one animal, data from age- and sex- matched mice displayed, line at median, and p value determined with one- length Mst1 and LPL were fused to N-Luc and C-Luc, respectively, way ANOVA. DIC, differential interference contrast. and expressed in 293T cells (Fig. 2A). Vimentin–C-Luc was used as a negative control, because vimentin and Mst1 have not been reported to interact. As a positive control, we used Mob1–C-Luc calponin-homology domains, each pair of which constitutes an because Mob1 is a known substrate of Mst1 (30). Coexpression of actin-binding domain. The sequence of the N-terminal domain Mst1–N-Luc with LPL–C-Luc resulted in increased levels of bio- contains a conserved phosphorylation sequence for Mst kinases luminescence over vimentin–C-Luc and C-Luc alone (Fig. 2B). In centered on Thr89 (Fig. 3B). A protein phosphorylation database fact, the bioluminescence resulting from LPL–C-Luc coexpression (http://www.phosphosite.org) reveals that Thr89 of LPL is phos- with Mst1–N-Luc was even greater than that from the positive phorylated in Jurkat T cells. Using a NetPhorest algorithm, we de- control of Mob1–C-Luc with Mst1–N-Luc (Fig. 2B). Thus, Mst1 termined that the probability of Mst1 phosphorylating LPL at Thr89 and LPL are able to interact, when coexpressed in 293T cells, (0.2339) was similar to the probabilities for phosphorylation for supporting the hypothesis that LPL may be a downstream effector Mob1b, a known Mst1 substrate, at residues Thr12 (0.2202) and of Mst1. Thr35 (0.1501). Of note, Thr89 is evolutionarily conserved across vertebrates (Supplemental Fig. 1), and it appears in all three Mst1 phosphorylation site in the LPL N-terminal domain plastin isoforms in mammals (Fig. 3B). The N-terminal domain of LPL has been described as a regulatory We therefore investigated whether Mst1 phosphorylates LPL. headpiece, containing a serine phosphorylation site at Ser5, two First, we asked whether Mst1 deficiency altered LPL posttransla- EF calcium-binding domains, and a calmodulin-binding site (20) tional modification. We observed increased mobility of LPL from (Fig. 3A). The C-terminal portion of LPL is composed of four Mst1-deficient cells during electrophoresis on SDS-polyacrylamide 1686 LPL IS A DOWNSTREAM EFFECTOR OF Mst1 IN T CELLS

or T89A LPL with LifeAct-Ruby (Addgene) in CD4+ T cells from LPL2/2 mice. Cells were stimulated with chemokine and then fixed for fluorescence microscopy. The signal from GFP was used to localize the expressed Wt or T89A LPL, and LifeAct-Ruby was used to visualize F-actin. Lamellipodia appeared as F-actin–rich flat protrusions at the leading edge of migrating cells, and they were apparent in cells that expressed Wt LPL or T89A LPL (Fig. 4A, solid arrows). Thus, phosphorylation of LPL at Thr89 appears to be dispensable for lamellipodial formation. A FIGURE 2. Mst1 and LPL interact in 293T cells. ( ) Schematic rep- We then analyzed the relative cellular distribution of Wt or T89A resentation of method to test Mst1–LPL interaction using luciferase re- LPL-GFP. Intensity profiles from projections along the indicated constitution assay. (B) Interactions between Mst1 and target proteins were evaluated by their ability to reconstitute luciferase activity. 293T cells were lines revealed a peak of Wt LPL-GFP intensity colocalizing with a cotransfected with Mst1 fused to N-Luc and the indicated targets fused to peak of F-actin intensity (Fig. 4A), suggesting that Wt LPL-GFP is C-Luc. Empty C-Luc and Vimentin–C-Luc fusion proteins were used as enriched in lamellipodia. However, intensity profiles from cells negative controls. C-Luc fused to Mob1a, a known Mst1 target, was used expressing T89A LPL-GFP revealed that the peak intensity of as a positive control. Data are representative of two to three independent GFP did not coincide with the peak intensity of lamellipodial experiments; p value determined using Mann–Whitney test. r.u., relative units. F-actin (Fig. 4A), suggesting diminished lamellipodial recruitment of T89A LPL. The percentage of cells with Wt LPL or T89A LPL gels compared with Wt cells, consistent with loss of a posttrans- enriched in the lamellipodia was quantified, demonstrating that Wt Downloaded from lational modification (Fig. 3C). Second, we evaluated the ability of LPL was more likely than T89A LPL to be enriched in the purified Mst1 to phosphorylate rLPL in vitro. Wt, but not kinase- lamellipodium (Fig. 4C). dead, Mst1 phosphorylated the isolated regulatory N-terminal domain We next examined the distribution of F-actin by LifeAct-Ruby of LPL, as measured by an in vitro kinase assay. We determined fluorescence (Addgene). Expression of Wt LPL-GFP led to Thr89 was the target of phosphorylation in vitro by observing a loss of lamellipodia with a smooth and dense distribution of F-actin in a 32 89 [ P] incorporation when the Thr in N-terminal domain of LPL was majority of cells (Fig. 4B, smooth). A smooth morphology of http://www.jimmunol.org/ changed to alanine (T89A mutant; Fig. 3D). Finally, we tested full- lamellipodial F-actin was defined as relatively even fluorescence length LPL as a substrate. Mst1 phosphorylated full-length Wt LPL intensity across the lamellipodia with few, if any, aggregates of but not T89A LPL when coexpressed in cells, based on immunoblots greater intensity. In contrast, more than half of cells expressing with an anti-phosphothreonine Ab (Fig. 3E). From these results, we T89A LPL-GFP displayed abnormal clumping of F-actin in or conclude that LPL Thr89 is a phosphorylation site for Mst1. near lamellipodia (Fig. 4A, white arrow; Fig. 4B, arrows). The clumped morphology was defined as irregularly shaped accumu- T89A LPL localizes poorly to lamellipodia and fails to restore lations of F-actin located at the border of lamellipodia and cell normal lamellipodial F-actin bodies, with fluorescence intensity greater than or equal to that of We next investigated the cellular function of phosphorylation of the lamellipodial F-actin. Quantification of the percentages of by guest on September 25, 2021 LPL Thr89. To determine if phosphorylation of LPL Thr89 affected cells exhibiting a clumped F-actin morphology by an independent F-actin morphology in T cells, we expressed GFP-tagged Wt LPL blinded observer revealed that the clumped morphology was more

FIGURE 3. Mst1 can phosphorylate the Thr89 residue in the N-terminal portion of LPL. (A) Schematic of LPL regulatory and actin-binding domains. The N-terminal regulatory domain contains a known phosphorylation site at Ser5 (S5; star), two EF-hands that generate a calcium binding site (EF), a cal- modulin binding site (CaM), and a putative Mst1-phosphorylation site centered on Thr89 (T89). The C-terminal domain contains four calponin homology (CH) domains, each pair of which creates an actin-binding domain. (B) Alignment of the portion of the regulatory domain of all three mouse plastins that contain the Mst1 phosphorylation site, by ClustalW. The asterisk (*) indicates identity and the colon (:) indicates similarity. (C) Immunoblot of LPL in whole-cell lysates of CD4+ T cells isolated from Wt and Mst1h/h mice, separated by SDS-PAGE. (D) Top panel, In vitro phosphorylation of Wt or T89A mutant LPL regulatory domain (residues 1–112) by Wt or kinase-defective (kd) Mst1 with [32P], visualized by autoradiography. Bottom panel, rLPL input proteins were visualized by Coomassie blue staining. (E) Mst1-mediated phosphorylation of full-length Wt or T89A mutant LPL visualized by immunoblot analysis of transfected 293T cell lysates with an Ab specific for phospho–Thr-X-Arg. The Journal of Immunology 1687 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Phosphorylation of Thr89 supports LPL localization to the lamellipod. (A and B)PurifiedCD4+ T cells from LPL2/2 mice were reconstituted with Wt or T89A LPL-GFP and with LifeAct-Ruby, stimulated with CCL19 in ICAM-1–coated chamberslides, fixed, and visualized by confocal microscopy. (A)Three representative cells are shown for Wt LPL-GFP and for T89A LPL-GFP. Intensity profiles of LifeAct-Ruby (filled, gray) and GFP (Wt LPL or T89A LPL; solid line) were calculated along the white lines indicated on the images. On the plots, the x-axisispixelnumbers,thelefty-axis is LifeAct-Ruby intensity (relative units), and the right y-axis indicates GFP intensity (relative units). An example of clumping is indicated by the white arrow. (B) Three representative cells are shown for Wt LPL-GFP and for T89A LPL-GFP. Arrows indicate clumped morphology of F-actin. Scale bars, 10 mm. (C) Confocal micrographs of cells expressing Wt or T89A LPL-GFP from (A)and(B) were blindly scored for GFP enrichment in the lamellipodia and for F-actin clumping. Data combined from three independent experiments; p value de- termined by Fisher exact test. (D) Alexa Fluor 488–conjugated rabbit muscle actin was polymerized prior to addition of kinase buffer only (buffer) or rLPL previously incubated with Mst1 (pLPL) or without Mst1 (LPL). Actin filaments and bundles were visualized by TIRF microscopy (Olympus Life Science). Scale bars, 10 mm. 1688 LPL IS A DOWNSTREAM EFFECTOR OF Mst1 IN T CELLS frequently observed in cells expressing T89A LPL (Fig. 4C). Thus, ablation of the putative Mst1 phosphorylation impaired both redistribution of LPL into the lamellipodia and disrupted normal lamellipodial actin morphology. Phosphorylation of Thr89 dispensable for actin-bundling activity Phosphorylation of Ser5 has been reported to enhance F-actin binding by LPL (21). We therefore asked if phosphorylation of LPL by Mst1 would affect actin-bundling activity, which results from F-actin binding. Purified rLPL was incubated with or without Mst1 in vitro and then added to pure actin filaments. Fluorescent actin (Alexa Fluor 488) was included to permit visualization of filament bundling by fluorescence microscopy. Actin filaments without LPL appeared as a diffuse network of thin filaments, as one would expect for single filaments that are not bundled (Fig. 4D). LPL not phosphorylated by Mst1 caused bundles of actin filaments to form, as did LPL that was incubated with Mst1 FIGURE 5. Thr89 phosphorylation is not required for interaction with

(Fig. 4D). The similar appearance of actin bundles indicates that CaM. Cell extracts containing GFP, Wt LPL-GFP, or T89A LPL–GFP Downloaded from Mst1 phosphorylation of LPL is not required for F-actin bundling fusion proteins were incubated with CaM-coated beads in the presence of activity. EGTA or 1 mM CaCl2. LPL binding to CaM was assessed by immunoblots with anti-GFP and anti-CaM. Data are from one experiment, representative Phosphorylation of Thr89 dispensable for calmodulin binding of four independent experiments. The Thr89 residue is found within a calmodulin (CaM) binding site

known to be important for the accumulation of LPL in the im- http://www.jimmunol.org/ munological synapse (23). To test whether defective CaM binding mature thymocytes, which express low levels of CD69 and CD24 was responsible for the effect of the T89A mutation observed in (19). In this study, thymic analysis of GFPneg (LPL-deficient) cells this study, we evaluated the binding of Wt and T89A LPL to CaM- from the bone marrow chimeras revealed the phenotypes previously coated beads (Fig. 5). The level of binding of Wt or T89A LPL to reported, with increased proportions of CD4SP (11–13%) and CaM-coated beads appeared to be similar, indicating that ablation CD8SP (2%), of which a substantial proportion were mature of the Mst1 phosphorylation site did not disrupt the CaM–LPL CD69negCD24neg SP thymocytes (Fig. 6C). interaction. Thus, phosphorylation of Thr89 does not appear to In mice reconstituted with Wt LPL-infected HSCs, the pop- regulated the ability of LPL to bind CaM. ulations of GFP+ thymocytes were more normal, with relative decreases in the populations of CD4SP (5.31% GFP+ Wt versus by guest on September 25, 2021 T89A LPL fails to fully rescue T cell migration in vitro and 13.3% GFPneg LPL-deficient) and CD8SP (0.84% GFP+ Wt versus in vivo 2.64% GFPneg LPL-deficient) thymocytes. The mature (CD69neg To determine whether phosphorylation of LPL Thr89 is required for CD24neg) population of SP thymocytes was decreased (38.5% of normal T cell migration, we compared CCL19-dependent migra- GFP+ CD4SP cells versus 63.7% of GFPneg CD4SP thymocytes), tion of LPL2/2 T cells reconstituted with Wt or T89A LPL–GFP also consistent with restoration of thymic egress. In contrast, re- in an in vitro transwell assay. Expression of Wt LPL-GFP resulted constitution with T89A LPL did not improve the thymic pheno- in substantial restoration of T cell migration compared with LPL- type of LPL2/2 mice. In fact, re-expression of T89A LPL resulted deficient T cells (Fig. 6A). In contrast, expression of T89A LPL- in increased accumulation of GFP+CD4SP (28.5% of GFP+ versus GFP provided partial restoration of migration (Fig. 6A). Wt LPL 11.4% of GFPneg) and GFP+CD8SP (18.8% of GFP+ versus 2.19% and T89A LPL were expressed at comparable levels, as detected of GFPneg) thymocytes. Additionally, the percentage of mature by flow cytometry. Thus, the Mst1 phosphorylation site was required (CD69negCD24neg) CD4SP thymocytes was increased in the GFP+ for full restoration of LPL functionality during T cell migration, as population (82% of GFP+ versus 42.5% of GFPneg), suggesting measured in vitro. greater impairment of thymic egress than observed for LPL defi- To evaluate the importance of Thr89 for cell migration in vivo, ciency alone (Fig. 6C). The failure of T89A LPL to rescue thymic we generated bone marrow chimeras expressing Wt or T89A LPL egress was not due to inefficiency of reconstitution, because sim- in LPL2/2 hematopoietic cells and assessed the phenotype of ilar phenotypes were obtained when Wt LPL and T89A LPL were thymic egress (Fig. 6B, 6C). Donor HSCs from LPL2/2 mice were expressed at comparable levels (Supplemental Fig. 2). Thus, we infected with lentivirus carrying plasmids for expression of Wt or found that re-expression of Wt LPL rescued the phenotype of T89A LPL-GFP and then injected into lethally irradiated congeni- impaired thymic egress in the LPL-deficient background, whereas cally marked CD45.1+ mice. Eight weeks postreconstitution, thy- re-expression of T89A LPL, which cannot be phosphorylated by mocytes derived from LPL2/2 HSCs were analyzed by flow cytometry Mst1, did not. We conclude that the Mst1 phosphorylation site (Fig. 6B). Lentiviral transduction of LPL2/2 HSCs is not 100% effi- Thr89 is required for normal T cell trafficking in vivo, as seen cient, so we observed a mixed population of donor-derived T cells in in vitro. recipient mice. The presence of GFP in cells indicated expression of In summary, we discovered that a phosphorylation site for Mst1 LPL, whereas GFP-negative cells were not infected and served as exists in the N-terminal regulatory domain of LPL and that ablation internal controls for LPL-deficiency. of this phosphorylation site impairs the ability of re-expressed LPL We examined several aspects of the thymic egress phenotype in to rescue lamellipodial LPL localization, F-actin lamellipodial these chimeric mice. LPL2/2 mice display increased proportions of morphology, and T cell migration in vitro and in vivo. Our evidence CD4SP and CD8SP thymocyte populations compared with Wt mice strongly suggests that LPL is a direct downstream effector of Mst1 (19). In addition, LPL2/2 mice display an abnormal accumulation of in promoting normal T cell migration. The Journal of Immunology 1689 Downloaded from http://www.jimmunol.org/

FIGURE 6. T89A LPL does not fully rescue T cell migration in vitro or in vivo. (A) CD4+ T cells purified from LPL2/2 mice were reconstituted with Wt or T89A LPL-GFP. The mixed population containing reconstituted (GFP+, Wt or T89A LPL) and nonreconstituted (GFPneg, LPL-deficient) cells were seeded into the top well of a 5-mm transwell with 100 ng/ml CCL19 in the bottom well. Cells were counted by flow cytometry. Activated CD4+ T cells from Wt mice are included as a positive control. Data are normalized to migratory efficiency of GFPneg (nonreconstituted, LPL-deficient) T cells and are representative of three independent experiments; p value determined by two-way ANOVA. (B) Schematic representation of generation of bone marrow + 2/2 2/2 chimeras. HSCs from CD45.2 LPL donors were infected with lentiviral constructs encoding Wt or T89A LPL-GFP in LPL HSCs and then by guest on September 25, 2021 transferred into irradiated CD45.1+ Wt recipients. Thymocytes were analyzed 8 wk after engraftment. (C) Thymic phenotype of bone marrow chimeric mice re-expressing Wt LPL or T89A LPL in an LPL2/2 background. Nonreconstituted LPL2/2 cells were identified as CD45.2+-GFPneg and Wt or T89A LPL-reconstituted cells were identified as CD45.2+-GFP+. Data are from one experiment, representative of two independent experiments.

Discussion at the border of the lamellipodia with the cell body and with F-actin T cell migration requires dynamic reorganization of the actin fluorescence intensity greater than or equal to that of the lamellipodia. cytoskeleton on a rapid time scale. Individual actin filaments as- In general, F-actin clumps colocalized with Wt or T89A LPL semble and disassemble, and actin filaments are linked into bundles (Fig. 4B). It is important to note that the clumped morphology also and networks. These processes combine to create higher-order struc- appeared in cells expressing Wt LPL, albeit at a significantly tures, with spatial and temporal regulation, to control cell morphology lower frequency, suggesting that the observed F-actin clumps do and polarity and to drive directional migration. The actin-bundling not represent a novel structure but simply a normal structure protein LPL is required for formation of lamellipodia (17) and for appearing at increased frequency in cells expressing T89A LPL. efficient T cell migration and thymic egress (19). Lamellipodia are highly dynamic structures, undergoing cycles of The most important discovery in this study is that the Mst1/Hippo extension and retraction that require rapid remodeling of F-actin kinase functions as a key upstream regulator to control LPL lo- and turnover of cell adhesion sites (31). We propose that actin and calization to the lamellipodia. We found that Mst1 interacted with LPL form aggregates at the junction of the cell body and the and phosphorylated LPL at Thr89. The nonphosphorylatable LPL lamellipodia either during lamellipodia extension or retraction. In mutant T89A was not able to rescue cell migration defects resulting this model, the inability to phosphorylate LPL on Thr89 prevents from LPL deficiency. We conclude that the Mst1 phosphorylation the normal rapid turnover of F-actin, locking the lamellipodia into site on LPL is functionally required for T cell migration. This novel one particular conformation and increasing the frequency at which regulatory pathway provides a mechanistic link between these two the F-actin clumps are visualized. Impaired lamellipodial dy- known components of the T cell migration machinery. namics would explain the reduced migration observed in T cells The effects of phosphorylation of LPL at Thr89 on the dynamic expressing T89A LPL. Future research will focus upon elucidating assembly of the actin-based structures involved in T cell migration the dynamics of F-actin aggregates in cells expressing T89A LPL. are an area of active investigation. Cells expressing T89A LPL LPL contains other regulatory elements, in addition to the Thr89 were still able to generate a leading edge, suggesting that phos- phosphorylation site. The LPL N-terminal domain contains a phorylation of Thr89 is not required for cell polarization or for serine phosphorylation site (Ser5), two EF-hand calcium-binding lamellipodial generation. However, we did identify a significant in- sites, and a calmodulin-binding site (Fig. 3A) (20). Ser5 is phos- crease in the proportion of T cells exhibiting a clumped morphology phorylated by protein kinase C isoforms, and this also correlates of F-actin. Clumps were defined as irregular accumulations of F-actin with translocation of LPL to lamellipodia (17, 21). However, a 1690 LPL IS A DOWNSTREAM EFFECTOR OF Mst1 IN T CELLS functional role for protein kinase C–mediated phosphorylation of 5. Nehme, N. T., J. Pachlopnik Schmid, F. Debeurme, I. Andre´-Schmutz, A. Lim, 5 P. Nitschke, F. Rieux-Laucat, P. Lutz, C. 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