The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells Dawang Zhoua,b, Benjamin D. Medoffc,d, Lanfen Chene, Lequn Lif, Xian-feng Zhanga,b, Maria Praskovaa,b, Matthew Liua,b, Aimee Landryg, Richard S. Blumberge, Vassiliki A. Boussiotisf,h, Ramnik Xavierg,i, and Joseph Avrucha,b,1 aDepartment of Molecular Biology and the bDiabetes, cPulmonary and Critical Care, iGastrointestinal, and hHematology and Oncology Units of the Medical Services, dCenter for Immunology and Inflammatory Diseases, gCenter for Computational and Integrative Biology and fDivision for Surgical Services, Cancer Center of the Massachusetts General Hospital; and eDivision of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02114 Communicated by John T. Potts Jr., Massachusetts General Hospital, Charlestown, MA, October 27, 2008 (received for review May 16, 2008) The Mst1 and Mst2 protein kinases are the mammalian homologs facilitated by the binding of both kinases to the noncatalytic of hippo, a major inhibitor of cell proliferation in Drosophila. Mst1 scaffold protein salvador/shar-pei; hippo also phosphorylates the is most abundant in lymphoid tissues. Mice lacking Mst1 exhibit noncatalytic protein MATS, which then binds to warts/LATS, markedly reduced levels of the Mst1 regulatory protein Nore1B/ promoting its autophosphorylation and activation. Activated RAPL in lymphoid cells, whereas Mst2 abundance is unaltered. warts, in turn, phosphorylates and negatively regulates the Mst1-null mice exhibit normal T cell development but low numbers transcriptional coactivator yorkie. Loss of hippo, warts/LATS, of mature naïve T cells with relatively normal numbers of effector/ Salvador, or MATS each results in overgrowth of a similar memory T cells. In vitro, the Mst1-deficient naïve T cells exhibit nature, whereas yorkie loss-of-function reduces growth and is markedly greater proliferation in response to stimulation of the T epistatic to the other components. Studies in mammalian sys- cell receptor whereas the proliferative responses of the Mst1-null tems indicate that this pathway is largely conserved; LATS1 (18) effector/memory T cell cohort is similar to wild type. Thus, elimi- and MOBKL1A/B (19), the mammalian orthologs of warts/ nation of Mst1 removes a barrier to the activation and proliferative LATS and MATS, are Mst1/2 substrates and negative regulators response of naïve T cells. The levels of Mst1 and Nore1B/RAPL in of cell proliferation (19, 20). Moreover, mice lacking LATS1 wild-type effector/memory T cells are approximately 10% those develop soft tissue sarcomas and ovarian tumors (21). Recipro- seen in wild-type naïve T cells, which may contribute to the cally, YAP, a mammalian homolog of yorkie, is phosphorylated enhanced proliferative responses of the former. Freshly isolated and negatively regulated by LATS1 (22–24). YAP is amplified in Mst1-null T cells exhibit high rates of ongoing apoptosis, a likely a wide variety of murine and human tumors (25); overexpression basis for their low numbers in vivo; they also exhibit defective of YAP transforms mammary epithelial cells in vitro (25) and clustering of LFA-1, as previously observed for Nore1B/RAPL- produces hyperplasia and cancer in mouse liver (22, 26). As deficient T cells. Among known Mst1 substrates, only the phos- regards other outputs, Mst1 activated by oxidant stress phos- phorylation of the cell cycle inhibitory proteins MOBKL1A/B is lost phorylates the Foxo1/3a polypeptides in their forkhead domain, CELL BIOLOGY entirely in TCR-stimulated, Mst1-deficient T cells. Mst1/2-catalyzed disrupting 14–3-3 binding and inhibiting the ability of Akt to MOBKL1A/B phosphorylation slows proliferation and is therefore promote Foxo1/3a nuclear exit (27). a likely contributor to the anti-proliferative action of Mst1 in naïve To define more fully the physiologic roles of Mst1, we gen- T cells. The Nore1B/RAPL-Mst1 complex is a negative regulator of erated mice lacking Mst1 polypeptide expression. naïve T cell proliferation. Results Mst1-Deficient Lymphoid Cells Exhibit Much Lower Nore1B Polypep- Mob1 ͉ Mst1 knockout ͉ RAPL ͉ hippo ͉ Stk4 tide but Unaltered Mst2. Mice lacking Mst1 polypeptide expression were generated from an ES cell line bearing a gene trap inserted st1 and Mst2 are closely related mammalian class II GC between the first and second exons (see supporting information Mprotein (ser/thr) kinases (1) whose overexpression induces (SI) Fig. S1). Mst1-deficient mice survive development, are apoptosis in many transformed cell lines through a combination fertile, and appear unremarkable. Mst1 is expressed at greatest of p53- and Jnk-dependent pathways (2–4). Regarding the abundance in lymphoid organs (Fig. 1A); lymphocytes from the physiological roles of these kinases, Kinashi and colleagues Mst1 trap/trap mice (henceforth called Mst1Ϫ/Ϫ) lack Mst1 demonstrated that the ability of ␣CD3/␣CD28 and chemokines polypeptide but show unaltered levels of Mst2 (Fig. 1B Left). to promote the Rap1-GTP-dependent integrin clustering nec- Mst1 can be coprecipitated with rassf family polypeptides (9); in essary for T cell adhesion and migration (5) is strongly depen- lymphocytes, the Rassf5 isoform Nore1B/RAPL, a Ras-Rap- dent on Mst1 (6), which is proposed to be activated through the binding protein, is a major Mst1 partner (6). Surprisingly, the Rap1-GTP recruitment of a Nore1B (also called RAPL)/Mst1 level of the Nore1B/RAPL polypeptide is greatly diminished in complex to the immunological synapse (7). The Mst1 sub- the Mst1-null lymphocytes (Fig. 1B Left) despite unaltered strate(s) that mediate this response is (are) not known. Mst1 and Mst2 are found in constitutive complexes with members of the Rassf polypeptide family (8, 9), at least two of which, RASSF1A Author contributions: D.Z., B.D.M., L.L., R.S.B., V.A.B., R.X., and J.A. designed research; D.Z., and Nore1A/RASSF5, are well established tumor suppressors B.D.M., L.C., L.L., M.L., A.L., and R.X. performed research; X.Z. and M.P. contributed new reagents/analytic tools; D.Z., B.D.M., L.C., L.L., R.S.B., V.A.B., R.X., and J.A. analyzed data; (10). Nevertheless, direct evidence in support of a tumor sup- and D.Z., B.D.M., R.X., and J.A. wrote the paper. pressor function for Mst1 and/or Mst2 is not yet available. The authors declare no conflict of interest. Mst1 and Mst2 resemble most closely the drosophila protein Freely available online through the PNAS open access option. kinase hippo and human Mst2 can complement hippo deficiency 1To whom correspondence should be addressed at: Department of Molecular Biology, (11). Loss of hippo function results in massive overgrowth of the Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114. E-mail: fly eye, due to an acceleration of cell proliferation together with [email protected]. a deficit in developmental apoptosis (11–14). The pathway This article contains supporting information online at www.pnas.org/cgi/content/full/ downstream of hippo has been extensively described (15–17). 0810773105/DCSupplemental. Hippo phosphorylates the protein kinase warts/LATS, which is © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810773105 PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20321–20326 Downloaded by guest on September 23, 2021 Peripheral blood Spleen A CD4+ A B 25 40 * CD4+/CD62L+ CD4+ * CD4+/ CD8+/ muscle CD8+/ 30 CD8+ CD62L+ CD62L+ CD8+ CD62L+ CD4+/ * CD4+/ * stomach intestine liver lung thymus skin heart * Skel marrow lymph node cortex cerebellum kidney spleen 20 CD44+ CD8+/ CD44+ CD8+/ -Mst1 * CD44+ * 10 * CD44+ -Mst2 0 % total cells % total lymphocytes 6 CD4+ CD4+ l) 14 -Nore1B 3.5 * * CD4+/ / CD4+/CD62L+ 12 3 3 * CD8+ CD62L+ CD8+/ 2.5 CD8+/ 10 * CD8+ CD4+/ * CD62L+ -Mob1 2 CD62L+ 8 * * CD8+/ 6 CD44+ * 1.5 CD4+/ CD8+/ * 4 CD44+ -Actin 1 CD44+ CD44+ 0.5 Total cells X 10 2 0 Total cells (x10 B spleen spleen C +/+ +/- -/- +/+ -/- vector + - - Flag-MST1-WT C 17.45 24.08 11.27 -Mst1 - + - * * Flag-MST1-K59R Mst1 MST1- -28s - - + 3 -Nore1B Nore1B Nore1B- + + + * WT -Mob1 -18s 2 58.25 -Flag 21.33 -Rassf1C 1 CD8 CD44 47.90 12.07 -Mst2 -Nore1B 7.37 -28s Mst1 -Actin -Actin 0 -18s CD4 mRNA (KO/WT) er null liv 32.98 lung colon heart 18.08 Fig. 1. Characterization of Mst1-null mice. (A) Expression of Mst1, Mst2 and intestine stomach CD4 CD62L some other relevant polypeptides in mouse tissues. Mst1/2, Nore1B, and Mob1 expression in mouse organs were analyzed by immunoblot and anti-actin Fig. 2. Peripheral T cell cohorts in wild-type and Mst1-deficient mice. (A)T served as a loading control. (B) Polypeptide and mRNA expression in spleen lymphocytes in the blood of wild-type and Mst1-null mice. The number of ϩ ϩ ϩ ϩ from wild-type and Mst1 gene trap mice. Left, Immunoblot of spleen extracts CD4 , CD8 , and naïve CD4 and CD8 T cells in the peripheral blood of for the polypeptides indicated. Right, Northern blot of splenic RNA. 18S and Mst1-null mice compared with wild-type mice (n ϭ 4 pairs of Mst1-null and 28S ribosomal RNAs serve as internal controls. (C) The Mst1 polypeptide wild-type littermates). Asterisks in this and all subsequent figures indicates a enhances the level of coexpressed Nore1B/RAPL. Vector encoding FLAG- P Ͻ 0.05 or lower calculated from a two-tailed Student t test. Black bars are tagged Nore1B was cotransfected in HEK293 cells with empty vector or vector Mst1ϩ/ϩ and white bars are Mst1Ϫ/Ϫ in this and all later figures. (B) T cell encoding FLAG-Mst1(WT) or FLAG-Mst1(K59R). An immunoblot of the ex- populations in the Mst1-null and wild-type spleen.