s2fieldsofstudy: Biology Medicine

text:
CD160 is essential for NK-mediated IFN-γ production

Tu et al. generated a novel CD160-deficient mouse and showed impaired NK cell–mediated tumor elimination and IFN-γ production. CD160+ NK cells are functionally distinct in secretion of IFN-γ from their CD160− NK cell counterparts.

NK cells play multiple roles during the innate immune response, reacting to a myriad of challenges, including pathogen-infected cells, transplanted allogeneic cells, and tumor cells (Moretta et al., 2002;Lanier, 2005). These responses are tightly regulated through multiple activating and inhibitory receptors. Several structurally distinct receptors have been implicated in activating effector functions, including NKp46, NKG2D, 2B4 (CD244), and CS1 (CRACC; Sentman et al., 2006;Marcenaro et al., 2011). Unlike these ubiquitously expressed NK receptors, the CD160 receptor is selectively expressed on the fraction of NK cells with the highest cytotoxic functions (Maïza et al., 1993).
CD160 is an immunoglobulin-like, glycosylphosphatidylinositol-anchored protein with homology to killer-cell immunoglobulin-like receptors (Agrawal et al., 1999). In addition to its association with effector function, CD160 was demonstrated to bind broadly to MHC class I molecules with low affinity, first in humans (Barakonyi et al., 2004) and later in mice (Maeda et al., 2005). A recent study, however, demonstrated that human CD160 binds to herpesvirus entry mediator (HVEM), a TNF family member, with much higher affinity than to MHC class I, and leads to suppressed T cell responses in vitro (Cai et al., 2008). Whether this high-affinity interaction exists in vivo and and what role it plays remains unclear.
HVEM has been shown to regulate both the innate and adaptive responses through its multiple binding partners, both as a ligand and as a receptor. Via B and T lymphocyte attenuator (BTLA) on T cells, the delivery of HVEM is largely inhibitory, controlling T cell effector responses Deppong et al., 2006) and the innate response (Sun et al., 2009). In contrast, signaling through HVEM activates T cells by LIGHT/TNFSF14 (Cheung et al., lymphoma model. The B16 melanoma model requires NK cells for early control of tumor growth (Kakuta et al., 2002). When inoculated subcutaneously with B16 tumor cells, CD160 / mice developed tumors of significantly larger sizes than WT mice (Fig. 2 A). To determine whether CD160 is required specifically by NK cells for control of tumor growth, we depleted NK cells before tumor inoculation by injection of NK1.1 mAb. After NK cell depletion, WT mice supported significantly faster growing tumors, whereas CD160 / mice depleted of NK cells had similar tumor growth kinetics to untreated mice (Fig. 2 A). These data suggest that CD160 is required for NK-mediated B16 tumor challenge.
As CD160 has been shown to be up-regulated on not only NK cells but also T cells after activation, we crossed CD160 / mice with RAG1 / mice to focus on the potential role of CD160 on NK cells in the absence of adaptive immune system. Similar to naive CD160 / mice, no apparent defects in total numbers of splenocytes or splenic NK cells were observed in naive CD160/RAG / mice (Fig. 2 B). However, B16 tumors grew significantly larger in CD160/RAG / mice, compared with RAG / controls ( Fig. 2 C), demonstrating that, in the absence of T cells, CD160 is still necessary for a robust antitumor immune response to B16 melanoma.
To further investigate the role of CD160 in the NK cell response to tumors, we adapted the RMA-S lymphoma line to our subcutaneous tumor model. Because RMA-S cells have a defect in MHC class I assembly, thus lacking surface MHC class I, they make suitable targets for NK cells both in vivo and in vitro (Kärre et al., 1986;Ljunggren et al., 1991;Smyth et al., 1998). RMA-S inoculated subcutaneously into CD160/ RAG / mice grew significantly faster and larger than RAG / controls, demonstrating that CD160-deficient NK cells were profoundly impaired in clearing RMA-S tumor cells in vivo ( Fig. 2 D). Furthermore, the difference in tumor growth was no longer observed in mice inoculated with the parental RMA lymphomas, which express intact MHC class I and therefore do not activate NK cells (Fig. 2 E). These data demonstrate that CD160 is specifically required for an optimal antitumor immune response primarily mediated by NK cells.
Because we had observed CD160's induction on NK cells after in vitro stimulation ( Fig. 1 B), we next determined whether CD160 were induced in response to in vivo tumor burdens. As such, we inoculated RMA-S tumors in WT or CD160 / mice, and harvested tumor tissues and splenocytes after the tumor-burden reached a designated size of 500 mm 3 . Consistent with our previous observation of CD160 induction after in vitro stimulation ( Fig. 1 B), we also observed an increase in CD160 expression on NK cells after in vivo stimulation in tumor-bearing mice compared with naive mice (Fig. 2 F). This suggests that CD160 may exert its role systemically, as represented by the spleen in response to a subcutaneous tumor.
CD160 is not required for NK-mediated cytotoxicity but required for cytokine production Despite having normal numbers of NK cells, CD160 / mice were susceptible to early outgrowing tumors. Therefore, we 2005; Cai and Freeman, 2009). However, the nature of the HVEM-class I MHC-CD160 interactions has not been well defined in vivo. To directly address these questions, we generated CD160 / mice and soluble CD160 (CD160-Ig) fusion protein and investigated the necessity and sufficiency of CD160 on the effector function of NK cells in vivo and in vitro. We reveal here that CD160 is a functional regulator of cytokine production by NK cells and is important for early control of tumor growth.

Generation of CD160-deficient mice
To define the role for CD160 in vivo, we generated a mouse strain on the C57BL/6 background with a targeted mutation of the CD160 gene ( Fig. 1 A). In this strain, exon 2, which contains the initiation codon and which is required for all known splice variants (Giustiniani et al., 2009), was replaced with a Neo cassette. Removal of exon 2 also rendered the downstream exons out of frame, ensuring the absence of any CD160 protein sequence. We confirmed by electrophoresis that no exon 2-containing CD160 transcripts existed in our KO strain, and that primers amplifying regions spanning exon 2 were the correct size for transcripts lacking this exon ( Fig. 1 A). The molecular weights for the WT and KO Southern bands were 11,183 and 8,408 bp, respectively. To verify the loss of CD160 protein expression in our CD160 / mouse, splenocytes from WT and CD160 / mice were labeled with fluorescence-coupled CD160 mAb or isotype control. Consistent with previous works (Maeda et al., 2005;Rabot et al., 2006Rabot et al., , 2007, resting NK cells from WT mice expressed limited amounts of surface CD160. However, when NK cells were stimulated with increasing concentrations of IL-2, the surface expression of CD160 was significantly elevated in an IL-2 dose-dependent manner within 18 h of stimulation, a response consistent with a previous study (Fig. 1 B;Le Bouteiller et al., 2002). In comparison, NK cells from the CD160-deficient mice had no detectable CD160 expression at all doses of IL-2 tested ( Fig. 1 B). This not only confirmed the lack of CD160 protein in genetically deficient mice, but also suggested that CD160 may play a regulatory role in NK activity.
Using this CD160 / strain, we determined whether CD160 deficiency alters the frequency of T cells, NK cells, or NKT cells, each of which have been previously reported to express CD160 (Agrawal et al., 1999;Le Bouteiller et al., 2002;Barakonyi et al., 2004;Maeda et al., 2005;Rabot et al., 2006;Cai et al., 2008). Total numbers of splenocytes, splenic T cells, NK cells, and NKT cells, as well as thymic T cell subsets were comparable between WT and CD160 / mice (Fig. 1, C and D). Therefore, it appears that CD160 does not play a significant role in the generation of T cells or NK cells.

CD160 is required for early control of NK-dependent tumors
To determine whether NK function is altered in the absence of CD160, we used two distinct NK-dependent tumor models: the B16 mouse melanoma model and the RMA-S (top) Relevant portion of WT mouse chromosome 3, targeting vector, and genomic sequence after homologous recombination. Exon 1, exon 2 (replaced by Neo sequence in KO), and DrdI and EcoRV restriction sites used for typing by Southern blot are shown. (bottom) 5 Southern blot verifying mutant gene. *, correctly targeted clones successfully used to make CD160 / founder lines. (right) Electrophoresis by Southern and Northern blots of CD160 exons from CD160 +/ and CD160 / splenocytes. The molecular weights for the WT and KO Southern bands were 11,183 and 8,408 bp, respectively. (B) Splenocytes from WT and CD160 / mice were cultured in complete medium supplemented with increasing concentrations of rhIL-2 (0, 100, 250, and 500 U/ml). After 18 h, cells were harvested and labeled with NK1.1, TCR, and CD160 mAbs. FACS histograms show NK1.1 + TCR  cells. Numbers in FACS plots indicate percentages of gated populations of WT cells. Splenocytes (C) or thymocytes (D) were harvested and labeled with TCR and NK1.1 or CD4 and CD8 mAbs, respectively. Cell populations were gated as shown in FACS plots and cell numbers were calculated from total cell counts. Data are representative of three independent experiments. evaluated the functional role of CD160 on NK cells. First, we evaluated whether NK cytotoxicity was impaired in CD160 / NK cells. We used the chromium release assay of YAC-1 target cells, which are highly sensitive to direct NK killing upon cell-to-cell contact with NK cells. We radiolabeled the target cells and co-cultured them with freshly isolated deficiency suggests that the mice in our tumor models may not be controlling tumor growth by means of cytotoxicity.
The activity of an NK cell is thought to depend on cumulative signals delivered by activating and inhibitory signals, splenocytes from naive RAG / and CD160/RAG / mice. We found no defect in cytotoxicity of CD160-deficient mice, indicating that CD160 is not required for NK-mediated cytotoxicity (Fig. 3 A). The intact killing activity despite CD160 Figure 2. CD160 is required for controlling NK-sensitive tumors. (A) WT and CD160 / mice were inoculated subcutaneously with 10 5 B16 melanoma cells. NK-depleted mice were administered NK1.1 mAb 1 d before tumor inoculation, and weekly thereafter. *, significant differences (P < 0.05) at days 17 and 20 between WT and CD160 / groups, and WT and WT (NK dep) groups. Differences between CD160 / and CD160 / (NK dep), and WT (NK dep) and CD160 / (NK dep) were not significant. Each group contained five mice. Data are representative of three independent experiments. (B) Splenocytes from RAG / and CD160/RAG / mice were labeled with NK1.1 and NKp46 mAbs. Cell populations were gated as shown in FACS plots and cell numbers were calculated from total cell counts. Data are representative of three independent experiments. (C) RAG / and CD160/RAG / mice were inoculated subcutaneously with 5 × 10 5 B16 melanoma cells. *, significant differences (P < 0.05) between groups at days 15 and 16. Each group contained 5 mice. Data are representative of three independent experiments. (D) For the RMA-S tumor model, RAG / and CD160/RAG / mice were inoculated subcutaneously with 5 × 10 5 RMA-S. *, significant differences (P < 0.05) between RMA-S groups at days 16, 18, and 20 by tumor volume. (E) 5 × 10 5 RMA-S or RMA lymphoma cells were inoculated subcutaneously as before, and weights of tumors resected at day 22 after inoculation were measured. *, significant difference (P < 0.05) between RMA-S groups at day 22 by tumor weight. (F) The expression of CD160 on NK cells from tumor-bearing mice was determined by flow cytometry. Shown are cells gated from the 7-AAD  , CD3  , NK1.1 + population. Mice were inoculated with 2 × 10 6 RMA-S tumor cells, and tumors were allowed to grow until 500 mm 3 before termination. Lymphocytes from the spleen were harvested into single-cell suspension, and labeled with NK1.1, TCR, and CD160 mAbs as previously described. Data are representative of three independent experiments. was also observed in our CD160/RAG / mice and could not be rescued by the addition of high doses of IL-2, as increasing the concentration of IL-2 even up to 250 U/ml in culture failed to restore the level of IFN- to that of controls (Fig. 3 C).
To confirm that the cytokine defect was specifically within the NK population, we performed intracellular cytokine staining on NK cells after in vivo priming and in vitro stimulation, which revealed reduced IFN- within the NK1.1 + population of lymphocytes (Fig. 3 D). These results suggested that NK cells do require CD160 for optimal IFN- production. Additionally, equal numbers of NK cells persisted throughout these activation assays, with no differences in viability with greater engagement of activating receptors favoring NK activation (Vivier et al., 2004). Cross-linking of the surface NK1.1 receptor with its specific mAbs results in activation of NK cells and increased IFN- production (Kim et al., 2010). Therefore, we next determined whether the cytokine response was impaired in NK cells from CD160-deficient mice. To this end, we stimulated splenocytes from WT or CD160 / mice plate-bound anti-NK1.1 mAb, and measured the level of IFN-. While WT splenocytes demonstrated strong IFN- production, CD160 / splenocytes demonstrated poor IFN- production upon NK1.1 cross-linking ( Fig. 3 B). This impaired IFN- response of NK cells in the absence of CD160 The percent cytotoxicity from 51 Cr release assay are shown for total splenocytes freshly isolated from naive RAG / or CD160/RAG / mice and cultured with radiolabeled YAC-1 target cells for 4 h at the indicated effectorto-target ratios. Data are representative of three independent experiments. (B) Splenocytes from naive WT and CD160 / mice were cultured at 2 × 10 5 cells per well in 96-well flat bottom plates with complete medium supplemented with rhIL-2 (100 U/ml) and plate-bound anti-NK1.1 at indicated concentrations. After 48 h, culture supernatants were harvested and IFN- concentration was determined. (C) Splenocytes from naive RAG / and CD160/RAG / mice were cultured as in B, with indicated concentrations of IL-2 and either plate-bound anti-NK1.1 or isotype control antibodies. After 48 h, culture supernatants were harvested and IFN- concentration was determined. (D) For intracellular cytokine staining, RAG / and CD160/RAG / mice were primed with intraperitoneal injections of poly(I:C) (100 µg) 18 h before splenocytes were harvested and cultured for 6 h in complete media containing Brefeldin-A. During this 6-h culture, cells received rhIL-2 (100 U/ml) and plate-bound anti-NK1.1 mAb (1 µg /ml). (left) Representative FACS plots; (right) quantification of the absolute numbers of IFN-producing NK cells. Intracellular cytokine staining was performed using IFN-, NK1.1, and NKp46 mAbs. *, P < 0.05. Each group contained five mice. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Each group contained splenocytes pooled from 5 naive mice. with cytokines rather than cytotoxicity. In addition, to assess whether CD160 was required for controlling metastasis, we used a melanoma model in which B16 tumor cells were delivered retro-orbitally into RAG / or CD160/RAG / mice, and lung tissues were observed for metastatic nodules. We observed significantly increased metastatic nodules in the CD160/RAG / recipients (Fig. 4 C). Together with the inability to rescue activation by IL-2 or NK1.1 engagement, our data suggests that CD160 regulates essential NK activation pathways, leading to the production of IFN-.

CD160 defines a novel cytokine-producing arm of NK cells
To determine whether CD160 could be a marker for the cytokine-producing NK cell subset, we used CD160 to divide the NK population from RAG / control mice into CD160 + versus CD160  subsets. When stimulated with increasing dosage of IL-2 together with plate-bound anti-NK1.1, the CD160 + subset produced significantly greater IFN- than the CD160-negative subset ( Fig. 5 A). This demarcation of IFN--producing capacity could also be elicited by in vivo stimulation of RAG / mice with poly(I:C) followed by in vitro stimulation with PMA/ ionomycin ( Fig. 5 B), indicating that CD160 functionally defines an NK subset endowed with IFN--producing capacity.
between RAG / and CD160/RAG / mice (unpublished data), suggesting that CD160 may be regulating the effector function rather than cell viability or exhaustion of NK cells. Finally, we used real-time PCR to determine IFN- expression, which does not require ex vivo stimulation. In splenocytes from naive mice, we found no impact of CD160 deficiency on IFN- expression (Fig. 4 A). However, in tumor-bearing or poly(I:C)-treated mice, CD160-deficient mice had significantly reduced expression of IFN- (Fig. 4 A). This suggested that CD160 is required for cytokine production in activated NK cells.
Finally, we asked whether the reduced IFN- in mice lacking CD160 could directly result in outgrowing tumors in vivo. We neutralized IFN- by administering a depleting anti-IFN- mAb alongside B16 tumor inoculation and weekly i.p. injections thereafter. As a result, mice receiving neutralizing mAb had significantly faster growing tumors compared with isotype control (Fig. 4 B). Additionally, by reducing the numbers of tumor cells inoculated, we observed that IFN--neutralized hosts had significantly higher rates of tumor engraftment (unpublished data). This suggests that IFN- is important for early control of tumors, likely by NK cells, and opens the possibility that NK cells can control tumors . RNA was isolated from splenocytes by TRIzol extraction, reverse transcribed into cDNA, and quantitated by real-time PCR for IFN- and HPRT. All data in this figure are representative of three independent experiments. (B) IFN- is required for early tumor control. Mice were inoculated with 5 × 10 5 B16 melanoma cells subcutaneously. These mice were also intraperitoneally administered the anti-IFN- mAb (XMG1.2). Antibody treatments were continued on a weekly basis during tumor growth. Tumor volumes were measured every 2 d. (C) To assess control of tumor metastasis, tumor cells were intravenously delivered into recipient mice. Naive RAG / and CD160/RAG / were retro-orbitally injected with 3 × 10 5 B16 melanoma cells. After 14 d, mice were sacrificed and lungs were harvested. Black spots in the lungs were counted and graphed. **, P < 0.005. All data presented here are representatives of three independent experiments.
RMA-S lymphoma model of RAG / mice. Analogous to the B16 response, the RMA-S tumors also grew faster and larger after CD160-Ig intratumoral administration (Fig. 6 C,  left). Correspondingly, the splenic IFN- response after NK activation was also reduced in both early and late stages of tumor burdens (Fig. 6 C, right). Finally, to determine whether CD160-Ig treatment could impact the tumor response in CD160/RAG / mice, we intratumorally administered the fusion protein as previously described alongside both control RAG / and CD160/RAG / mice (unpublished data). Although CD160-Ig treatment compromises tumor control in RAG / mice, the treatment did not alter the tumor growth of CD160/RAG / mice, suggesting that CD160-Ig functions as a blocking rather than activating molecule in vivo. These data demonstrate that targeting the CD160 pathway in the otherwise unremarkable RAG / host could impair tumor control and IFN- production.
However, these experiments testing CD160-Ig used tumor-bearing mice and smaller tumor burden in treated group might influence the result. To determine whether CD160-Ig could also impair cytokine production independently of tumor burden, we administered CD160-Ig i.p. together with poly(I:C). Poly(I:C) was used to prime NK cells because such priming is required for ex vivo NK function and CD160-Ig had no effect over isotype control in naive mice (unpublished data). When we measured cytokine production 18 h after treatment, we observed significantly reduced IFN- production in CD160-Ig treated mice after ex vivo stimulation by plate-bound anti-NK1.1 and IL-2, as well as in freshly isolated splenocytes without additional stimulation (Fig. 6 D). These data further confirm that CD160 functionally regulates cytokine production in activated NK cells.

CD160 is intrinsically required by NK cells for efficient cytokine production
CD160 is required by NK cells for efficient production of IFN-. To determine whether this requirement occurs through a cell-intrinsic or -extrinsic mechanism, we generated mixed BM chimeric mice. We reconstituted lethally irradiated WT (CD45.1) hosts with equal ratios of WT (CD45.1) and CD160 / (CD45.2) BM cells. After reconstitution, we evaluated IFN- production as before, using poly(I:C) to prime the chimeric mice before ex vivo stimulation with PMA/ionomycin. We did not observe preferentially reduced NK numbers or percentage in the CD45.2 fraction over the CD45.1, further confirming that CD160 deficiency does not significantly impair NK development. However, intracellular cytokine staining revealed that the CD45.2 + CD160 / NK cells produced significantly lower IFN- than CD45.1 + WT NK cells even in the same recipients. Dividing the IFN- producers into low or high fractions, we further observed the reduction in the CD160 / NK cells consistently across both fractions (Fig. 7 A). In addition to a 1:1 chimera, we generated a chimera which was reconstituted with primarily (90%) CD160 / BM. In these chimera, we also observed that CD160 / NK cells produced even less IFN- per cell despite outnumbering the WT To determine whether the IFN--producing NK subset delineated by CD160 was important for tumor control and to evaluate its therapeutic potential, we transferred purified populations of CD160-expressing NK cells versus CD160 nonexpressing NK cells intratumorally into RMA-S tumors of CD160 / mice. To achieve clear separation of CD160 + versus CD160  NK populations, donor mice were first primed with poly(I:C), as previously described. The CD160 / mice regained control of tumor growth soon after receiving CD160 + NK cells compared with CD160 / mice receiving CD160  NK cells (Fig. 5 C). This indicates that the NK subset defined by CD160 is not only functional in vitro, but also helps control tumors in vivo and demonstrates the therapeutic potential of CD160 in the treatment of cancer.
Because CD160 expression can be induced after NK activation, we determined whether the NK subset defined by CD160 could maintain stable expression of CD160, and also whether the CD160-negative NK cells could induce new CD160 expression. After NK activation, the CD160 + fraction of NK cells maintained CD160 expression (Fig. 5 D). However, the CD160  fraction acquired CD160 expression, and began expressing significant levels comparable to the initially positive fractions by 6 d after culture. Interestingly, before day 4 of stimulation, there was no detectable expression of CD160 in the CD160  fraction. This suggests a degree of stability to CD160 expression, particularly for our in vitro assays evaluating CD160 + and CD160  NK fractions. However, after prolonged stimulation, NK cells can acquire new expression of CD160.

CD160 functionally regulates the NK response
Although absolute numbers of NK cells were not affected by CD160 deficiency, the possibility still exists that the defect seen in CD160-deficient mice in vivo could have been caused by issues of NK cell development or homeostasis. As such, to avoid the possible effect of CD160 during NK development, we generated a murine CD160-Ig fusion protein to functionally block CD160 interactions in vivo, serving as a soluble decoy receptor to CD160's natural ligands. Our CD160-Ig contained the mature extracellular CD160 sequence with mammalian posttranslational modifications, linked with the Fcportion of human IgG1.
To functionally target the CD160 pathway, we administered a low dose of CD160-Ig intratumorally in RAG / tumor-bearing mice as soon as tumors reached >75 mm 3 in size. The CD160-Ig-treated tumors grew larger than isotype control-treated tumors (Fig. 6 A, left). Splenocytes harvested from CD160-Ig-treated B16 melanoma-bearing mice were also significantly impaired for IFN- production after NK1.1 activation ex vivo (Fig. 6 A, right). To reduce the potential bias from differential tumor sizes between CD160-Ig and isotypetreated groups, we also determined the IFN- response relatively early after tumor inoculation, before the tumor sizes diverged (Fig. 6 B, left). Similarly between both early and late stages of tumor burden, we saw marked reduction in levels of IFN- after ex vivo NK activation (Fig. 6 B, right). Similar defects were observed when CD160-Ig was used in the To obtain pure populations of CD160 + versus CD160  NK cells, splenocytes freshly isolated from naive RAG / mice were sorted by flow cytometry for NK1.1 and CD160 expression into two fractions: NK cells expressing or lacking CD160. The two NK fractions were stimulated with increasing dosages of IL-2 together with plate-bound anti-NK1.1 (1 µg /ml), and supernatants were measured for cytokines after 48 h in culture. (B) We determined the cytokine production capacity of these subsets by priming mice with poly(I:C) for 18 h before harvesting splenocytes and restimulating the NK with PMA/ionomycin. Splenocytes were surface stained for NK1.1 and CD160, and fixed/permeabilized to stain IFN- intracellularly. (C) Donor RAG / mice were primed with poly(I:C) (100 µg, i.p.) for 18 h before harvesting of splenocytes, which were then stained for NK1.1 and CD160 for sorting. Sorted fractions of CD160expressing or CD160-non expressing NK cells were intratumorally injected into RMA-S tumors of CD160 / mice at 5 × 10 5 cells per 100 mm 3 -sized tumors. Tumor sizes were measured every 2 d. (D) To determine the stability of CD160 expression on CD160 + versus CD160  NK cells, WT and CD160 / naive mice were sacrificed, and freshly isolated splenocytes sorted by FACS for CD3  NK1.1 + CD160  and CD3  NK1.1 + CD160 + populations. CD160 + and CD160  populations from both WT and CD160 / donors were cultured with IL-2 (500 U/ml) for 2, 4, and 6 d. At each time point, cells were harvested, washed, and stained for CD160 expression. Representative FACS plots are shown to indicate CD160 expression levels over the 6 d of culture. Data are representative of three independent experiments. NK cells by almost 10-fold (Fig. 7 B). Together, these results demonstrate that CD160 is required intrinsically by NK cells for efficient IFN- production.

NK-CD160 coordinates with non-NK-HVEM to regulate IFN-
The binding partner to CD160 has remained elusive due in part to the promiscuous arrangement of competing binding partners. CD160 was first shown to bind to classical and nonclassical MHC class I molecules (Maeda et al., 2005), and was later shown to bind to HVEM with higher affinity (Cai et al., 2008). We have no evidence that CD160-Ig binds physiologically to class I molecules in vivo. To test whether CD160 can bind to HVEM, we used our CD160-Ig reagent, and demonstrated binding of CD160-Ig to 293-HEK cells transfected with HVEM (Fig. 8 A). To further confirm that CD160 interacts with HVEM, we developed an ELISA-based approach using HVEM-Ig and CD160-Ig fusion proteins. We demonstrated CD160's interaction with HVEM, using platebound mouse HVEM-Ig to capture soluble CD160-Ig. CD160-Ig was bound in a dose-dependent manner over isotype control (Fig. 8 B). Together, these data suggest that CD160 directly engages HVEM.
Because we demonstrated that CD160 is required on the NK cell, we next sought to determine whether HVEM is required on a partner cell. Because our methods for eliciting IFN- required poly(I:C) priming in vivo, we tested whether the DC or macrophage which express TLR-3 could provide the HVEM to engage CD160 on the NK cell. Consistent with RNA data from the public ImmGen database, we also observed that HVEM is most highly expressed on DCs and macrophages (unpublished data). As such, we developed an in vitro co-culture system to combine purified NK cells with DC/macrophages cultured from BM cells grown in GM-CSF. To determine whether HVEM on DC/macrophages were required to induce CD160-dependent IFN-, we co-cultured RAG / or CD160/RAG / purified NK cells together with WT or HVEM / BM-derived DC/macrophage. We observed that HVEM was required on the DC/macrophage as NK cells co-cultured with HVEM / DC/macrophage produced significantly less IFN- than WT controls (Fig. 8, C and D). These data, together with our in vivo poly(I:C) primed data suggest that CD160 regulates NK function through coordinating with HVEM on DC/macrophage accessory cells. which was at day 10 post-inoculation, followed by harvesting of splenocytes for NK activation to measure the IFN- response. (C) The RMA-S lymphoma model was used instead of B16, and the experiment was performed analogously to Figure 6A. (D) Naive RAG / mice were administered CD160-Ig or hIgG isotype control (50 µg) together with poly(I:C) (50 µg) intraperitoneally. After 18 h, spleens were harvested and stimulated ex vivo by plate-bound anti-NK1.1 and IL-2 as described before. Splenocytes were also analyzed by RT-PCR as described before. All data shown are each representative of 3 independent experiments. ***, P < 0.01.
we also showed that soluble CD160 could reduce the cytokine production by NK cells. Furthermore, we demonstrated that this cytokine response is an essential component of the innate response to control tumor growth. This was unexpected because cytotoxicity has long been considered the primary effector mechanism of NK cells (Trinchieri, 1989;Moretta et al., 2001Moretta et al., , 2002Yokoyama and Plougastel, 2003;Yokoyama, 2005). As such, our newly demonstrated functional role of CD160 highlights the cytokine arm of killer lymphocytes.
Our experiments demonstrated that CD160 was required for efficient IFN- production in activated NK cells, whereas resting NK cells were not impacted by CD160 deficiency. This suggested that CD160 functions as a co-stimulatory molecule for promoting NK cytokine production after a primary stimulus, such as poly(I:C) priming or NK1.1 cross-linking. One potential mechanism could be through receptor synergy. In our NK activation assays, we used plate-bound anti-NK1.1 mAb to agonize the NK1.1 receptor, which signals through the FcR DISCUSSION CD160 was first identified in a cytotoxicity screen to select lymphocytes with the highest cytotoxic capacity from human peripheral blood (Maïza et al., 1993), and has served as a marker for selecting NK cells and CTLs that can kill target cells more efficiently than their CD160  brethren (Barakonyi et al., 2004;Maeda et al., 2005;Fons et al., 2006;Tsujimura et al., 2006;Cai et al., 2008). Although this correlation with killing suggests that CD160 might play a stimulatory role, the engagement of CD160 by HVEM was shown to suppress the T cell response, raising the possibility that CD160 could also have inhibitory roles (Cai et al., 2008). Whether CD160 functionally regulates the cytotoxic lymphocytes has remained inconclusive, in part because of the lack of a genetic model. Here, we demonstrate that genetic deficiency of CD160 impaired the function of NK cells to produce cytokines, indicating that CD160 is functionally important for a specific arm of the NK effector function. Alongside the knockout model,

Figure 7. CD160 is required intrinsically by NK cells for IFN- production. (A)
To determine whether CD160 regulates NK cells intrinsically or extrinsically, we generated mixed BM chimeric mice. We used the C57BL/6 strain of mice bearing the congenic marker CD45.1 to demarcate WT hematopoetic cells from the CD45.2 expressing CD160 / cells. BM (2 × 10 6 cells) from WT (CD45.1) and CD160 / (CD45.2) donors were transferred at an equal ratio (1:1) into lethally irradiated (1,000 Rad) WT (CD45.1) recipients. Validation of chimerism was performed by staining peripheral blood lymphocytes. After 6 wk of reconstitution, mice were sacrificed and splenocytes were stimulated in vitro by PMA/ionomycin for intracellular cytokine staining. (A) The percentage of IFN--producing cells are quantitated in the histograms, with representative FACS plots below, showing the IFN- staining gated on either CD45.1 + or CD45.2 + CD3  NK1.1 + cells. The fraction of IFN- + NK cells were further divided into low versus high, as quantitated in the histograms above. (B) In addition to chimeras with equal ratio of hematopoietic grafts, we generated chimeras in which the dominant hematopoietic source was deficient of CD160 (90% CD160 / , 10% WT). As in A, histograms quantitating the percentage of IFN--producing NK cells are above the representative FACS plots. *, P < 0.05; ***, P < 0.0005. Data are representative of three independent experiments. NK-to-NK homotypic interactions, in which CD160  NK cells might provide more accessible HVEM than CD160 + NK cells because BTLA and CD160 has been recently demonstrated to bind to HVEM in cis (Cheung et al., 2009). It has recently been shown that CD160 competes with BTLA for binding to HVEM (Kojima et. al., 2011). In addition, poly(I:C) has broad immunological effects that extend beyond the DC. As such, the possibility of stromal cell involvement remains to be determined. Finally, the tumor itself could provide ligands to CD160. However, we demonstrated CD160's function on both B16 and RMA-S tumors, which rules out tumorderived MHC-I (because RMA-S lack surface MHC-I) as well as HVEM (because B16 lack surface HVEM).
To determine the mechanism underlying the overgrowing tumors in our CD160-deficient mice, we first assessed the cytotoxic capacity of the NK cell. To our surprise, CD160 was not required for killing because CD160-deficient NK cells could kill as well as CD160-sufficient NK cells. This was unexpected because CD160 expression was previously correlated with cytotoxicity (Agrawal et al., 1999;Le Bouteiller et al., 2002). In addition, earlier studies also showed that the capacity of an NK cell to produce cytokines was mutually exclusive with cytotoxicity (Yokoyama, 2005;Blasius et al., 2007). This suggested that CD160 might regulate a select subset of NK cells that is functionally distinct from other CD160  NK cells. As such, when we examined the stability of the NK fraction defined by CD160, we found that CD160 + NK cells remained positive, whereas CD160  NK cells gradually became positive after prolonged activation. Furthermore, the CD160 + NK fraction was endowed with greater cytokine production capacity and could rescue tumors in CD160 / hosts, compared with their CD160  brethren. Together, this data suggests that CD160 does not globally regulate the NK compartment, but instead exclusively regulates a distinct subset specialized for cytokine production.
CD160 is required for optimal IFN- production by NK cells, an underappreciated but critical mechanism for controlling tumors, which we confirmed through IFN- depletion in tumor-bearing mice. There are several possibilities as to how the NK cytokine response can control tumors. One possibility is that IFN- can inhibit the tumor cell cycle, and recent studies have shown that IFN- synergizes with TNF through TNFR1 expressed on tumor cells to induce cell cycle arrest (Kakuta et al., 2002;Müller-Hermelink et al., 2008;Braumüller et al., 2013). A second possibility is that the IFN--producing population of NK cells, which express CD160, could directly interact with tumor stroma, as was recently reported in human tumors in which mAb's against CD160 was able to inhibit angiogenesis (Fons et al., 2006). Finally, a third possibility is that the CD160 + subset of NK cells produce sufficient cytokines, such as IFN-, to activate other NK cells, lymphocytes, macrophages, and neutrophils to work together at controlling the tumor. This last avenue is highly probable given our finding that CD160 on NK cells may coordinate with HVEM on non-NK cells such as macrophages, which infiltrate the tumor microenvironment over the natural for its signal transduction possibly involving PKC- and CD45, which were shown to be essential in triggering transcription of IFN- without affecting the cytotoxicity. Similarly, cross-linking CD160 was shown to promote Akt and PI3K phosphorylation (Rabot et al., 2007;Liu et al., 2010). Because NK1.1 is enriched at lipid rafts (Fassett et al., 2001) and CD160, as a GPIanchored protein, is likely also enriched at lipid rafts (Rabot et al., 2007), CD160 could be converging with NK1.1 to enhance the downstream FcR, PKC--CD45, and Akt-PI3K pathways to promote IFN- production. (Arase et al., 1997;Vivier et al., 2004;Vahlne et al., 2008). Therefore, in the absence of CD160, the primary NK1.1-mediated stimulus to the NK cell might be less efficient, resulting in less IFN-.
Interestingly, given the recent reports that PKC- and CD45 tyrosine phosphatase impacts IFN- secretion but not cytotoxicity in NK cells, CD160 may preferentially signal through PKC- and/or CD45 (Tassi et al., 2008, Hesslein et al., 2006 downstream of NK1.1 activation to induce transcription of IFN-. This model of lipid raft-mediated receptor synergy is consistent with the current view of NK regulation, i.e., that NK cells depend on cumulative signals from activating and inhibitory receptors (Vivier et al., 2004). Based on this model, when an NK cell engages a target, receptor synergy is important for determining the outcome. Additionally, the model of receptor synergy is also supported by our demonstration that CD160 is required intrinsically by the NK cell. Although the other binding partner to HVEM, BTLA, has been shown to signal bi-directionally as both a receptor and ligand, our mixed BM chimeras showed that CD160 functions as an NK receptor, but not as a ligand, because WT NK cells that expressed CD160 were not able to rescue CD160 / NK cells within the same host. As such, this supports a revised model in which CD160 is a receptor on NK cells that receives signals from the widely expressed molecules HVEM and/or MHC-I. Additionally, whereas BTLA is expressed on human NK cells, we and others have observed significantly reduced BTLA expression on mouse NK cells (Han et al., 2004;Hurchla et al., 2005;Iwata et al., 2010;D'Addio et al., 2013). This discrepancy between mouse and human BTLA expression helps elucidate the role of CD160 in our mouse model by removing one potential binding partner on our NK cells.
By priming the mice with poly(I:C), which indirectly stimulates NK cells by first activating DCs through TLR-3, we observed reduced IFN- in CD160 / NK cells. This suggests that CD160 on NK cells could coordinate with DCs, which express both MHC-I and HVEM, the putative binding partners to CD160. In this regard, we showed for the first time that HVEM is required on the DC/macrophage compartment by NK cells for poly(I:C)-induced cytokine production. Although other cell types, including NK cells could also provide MHC-I and/or HVEM, we predict that the DC/ macrophage compartment is the main partner to CD160 + NK cells because they express the highest levels of surface HVEM, as well as innate receptors such as TLR-3 that respond to poly(I:C). However, this does not rule out the possibility of together are balanced by HVEM, not to mention the therapeutic potential of CD160 for fighting cancer.

MATERIALS AND METHODS
Animals. WT and RAG1 / C57BL/6 mice were purchased from Harlan Teklad. HVEM / C57BL/6 mice were described previously (Wang et al., 2005), and were backcrossed onto C57BL/6 background 10 generations, and then further crossed with RAG1 / mice to generate HVEM/RAG / . CD160 / mice were generated on the C57BL/6 background at the University of Chicago (Chicago, IL). A targeting vector containing a neo cassette flanked by homology arms to mouse chromosome 3 sequences surrounding CD160 exon 2 was generated by recombineering ( Fig. 1 A). This vector was introduced into C57BL/6-derived embryonic stem (ES) cell line PRX-B6N #1 (Primogenix, Inc.) and ES cell clones were screened by PCR and Southern blot to identify correctly recombined clones. 4-cell embryos from CD-1 mice were injected with 6-8 recombined ES cells to produce a mouse completely derived from the C57BL/6 ES cells. The resulting male mouse was mated with C57BL/6J females to produce CD160 +/ mice with germline transmission of the knockout allele. These mice were bred to homozygosity and the loss of CD160 transcription was verified by reverse-transcription PCR (rt-PCR) and protein expression by flow cytometry (Fig. 1, A and B). Primers used for rt-PCR were as course of tumor development. Furthermore, a recent study has demonstrated that CD160 demarcates a unique population of intraepithelial lymphocytes (currently classified as ILC-1) that specializes in producing IFN- (Fuchs et al., 2013). Thus, the role of CD160 on the host production of IFN- extends from the spleen to the gut. Whether and how the gut-derived IFN- coordinates with splenic-derived (and other tissue-derived) IFN- remains to be explored. As such, our study highlights CD160 and HVEM as a new immunological bridge in innate immunity that coordinates NK cells with non-NK cells.
Our animal model opens new possibilities to dissect this complex regulatory network, and establishes a much needed tool to probe how HVEM and CD160 together coordinate the immune response. Our demonstration of CD160's positive regulation of NK cells is unexpected, considering the previously reported inhibitory role of CD160 on T cells (Cai et al., 2008). Understanding this positive axis of CD160 is pivotal for understanding not only how the NK response is regulated, but also how the innate and adaptive responses The 293-HEK cell line was transiently transfected with an HVEM-expression plasmid using the transfection reagent polyethylenimine (PEI). Surface expression of HVEM was verified by the anti-HVEM mAb (HMHV-1B18). CD160-Ig was biotinylated and tetramized with streptavidin conjugated with the APC fluorophore, and the fusion protein tetramers were used to stain HVEM-transfected 293-HEK cells. (B, top) schematic of ELISA. Platebound mHVEM-mIg was incubated with mCD160-hIg and detected with anti-human IgG. (bottom) Adsorbed mHVEM-mIg was incubated with titered, purified mCD160-hIg or control hIgG isotype control. (C) To determine whether HVEM was required on DCs to trigger CD160-dependent IFN- production on NK cells, we co-cultured purified NK cells together with BM-derived DCs at a ratio of 1:0.3 of NK-to-DCs. Co-cultures were stimulated with poly(I:C) (100 µg/ml) for 48 h in round-bottom wells, after which supernatants were harvested for detection of cytokines by cytokine bead array. (D) We increased the cell densities while maintaining the same NK/DC ratio as before. The stimulation and measurement proceeded as previously described. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Data are representative of three independent experiments.
Flow cytometry, antibodies, and fusion protein. Flow cytometry analysis was performed on FACSCalibur, FACSCanto, and FACSAria II instruments (BD) and analyzed using FlowJo software (Tree Star). Antibodies were purchased from BD, BioLegend, Invitrogen, or eBioscience. Intracellular cytokine staining was performed using permeabilization and fixation buffers supplemented with saponin (eBioscience).
CD160-Ig fusion protein was created by joining the extracellular portion of mouse CD160 with the Fc-portion of human IgG1. The DNA sequence encoding expressed CD160 was determined based on the CD160 mature protein sequence. The plasmid containing this sequence was then cloned into a plasmid containing the human IgG1 cassette. Together, the mouse CD160 and human IgG1 plasmid was subcloned into a plasmid containing an Ig-Kappa leader sequence. Finally, Chinese hamster ovary cells were transfected with the final plasmid for protein production.
For fusion protein tetramerization, we first biotinylated the CD-Ig fusion protein or isotype control using the EZ-Link Sulfo-NHS-LC-Biotinylation kit according to the manufacturer's protocol (Pierce). Then, we multimerized the biotinylated monomers according to a previously published protocol (Altman and Davis, 2003).

Statistics.
Comparisons of data were analyzed by two-tailed Student's t test using GraphPad Prism 5.0. Data from such experiments are presented as mean values ± SEM. Differences of P < 0.05 were considered significant.
Cell lines. NK cells were obtained from spleen cell suspensions via negative enrichment, according to the manufacturer's protocol (Miltenyi Biotec). Enriched NK cells were cultured in complete RPMI with indicated concentrations of rhIL-2 (Chiron).
The B16/F10 murine metastatic melanoma cell line was grown on tissue culture plates, using complete RPMI media supplemented with 10% FBS. RMA (MHC class I H-2b positive) and RMA-S (MHC class I H-2b negative) thymoma cells were grown in suspension in complete RPMI supplemented with 10% FBS.
Tumor models. Mice were injected subcutaneously with tumor cells in the lower back. Tumor masses were measured by a millimeter caliper. Tumor volume was calculated by multiplying the width, length, and height of the tumor mass and dividing the product by two.
NK in vitro assays. NK cells were harvested from spleens and cultured in 96-well flat bottom tissue culture plates. Anti-NK1.1 (PK136) mAbs at the indicated concentrations were coated onto plates 1 d before NK cell culture (Kim et al., 2010). Titrations of IL-2 were added to complete RPMI medium containing 10% FBS.
Lymphocyte depletion. Lymphocytes were depleted by intraperitoneal injection of 200 µg of NK1.1 mAb in 100 µl of PBS. Antibodies were injected 1 d before the start of the tumor inoculation, as well as weekly thereafter for 2 wk, resulting in a total of 3 injections per mouse.
Cytometric bead array (CBA). Cytokine measurement was performed with the mouse inflammation CBA kit (BD) according to the manufacturer's guidelines.
Quantitative real-time PCR. Total RNA from cells were isolated with TRIzol reagent (Invitrogen) and treated with DNase I, followed by reverse transcription with random primers and AMV reverse transcription (Promega). Real-time RT-PCR was then performed with the SsoFast EvaGreen supermix (Bio-Rad Laboratories) on the StepOne Plus (Applied Biosystems).
Cytotoxicity assay. To measure direct NK-mediated cytotoxicity, we used the standard chromium 51 ( 51 Cr). Freshly isolated splenocytes from RAG / mice were lysed of RBCs, counted, and cultured with YAC-1 target tumor cells. The YAC-1 cells were labeled with 0.75 MBq/ml (20 µCi/ml) for 1 h at 37°C. Effector and target cells were diluted in RPMI-1640 media containing 10% FBS and Hepes buffer and incubated in round-bottom wells for 4 h at 37°C. Spontaneous 51 Cr release was determined by treating YAC-1 cells with media alone, and maximum release was determined by treating YAC-1 cells with Triton X-100 at 2.0% in media. Supernatants were harvested and


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1BS421A mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1BD239A mutant exhibited very low levels of p-S421 in cells but this was increased by KRASG12V. In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity. Electronic supplementary material The online version of this article (doi:10.1007/s00018-015-2032-x) contains supplementary material, which is available to authorized users.

Abstract The dual-specificity tyrosine-phosphorylationregulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B S421A mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B D239A mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS G12V . In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

Introduction
The dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are an evolutionarily conserved family of protein kinases that are found within the CMGC group (CDK, MAPK, GSK and CLK families) of the eukaryote kinome [1][2][3][4]. The mammalian DYRKs are divided into class I (DYRK1A and DYRK1B) and class II (DYRK2, DYRK3 and DYRK4) based on sequence homologies. The DYRKs require phosphorylation of the second tyrosine in the characteristic tyrosine-X-tyrosine (Y-X-Y) motif within their activation loop for kinase activity [5,6]. In contrast to many other kinases, this activation loop phosphorylation is not catalysed by an upstream, 'activating kinase' but occurs during translation when DYRKs undergo intramolecular cis-autophosphorylation on tyrosine [7]. As co-translational activation loop tyrosine phosphorylation is a one-time-only event, it was proposed that once synthesised, the mature DYRKs lose tyrosine kinase activity and phosphorylate substrates exclusively on serine or threonine residues. However, recent studies have demonstrated that mature DYRK1A retains the ability to autophosphorylate on other tyrosine residues outside the activation loop [8]. Regardless, substrate phosphorylation appears to occur exclusively on serine or threonine, usually within a proline-directed context (pS-P or pT-P) [9,10]. The DYRKs have roles in controlling transcription, mRNA splicing, cell cycle progression, survival and differentiation [2,3]. For example, loss of a single copy of Dyrk1a in mice leads to increased apoptosis and decreased brain size in mice [11]. In addition, the DYRK1A gene is situated in the Down's syndrome (DS) critical region on chromosome 21 (21q22. 1-22.3), is over-expressed in both foetal and adult brains of DS individuals [12] and is thought to contribute to the clinical features of Down's syndrome [13][14][15]. Indeed, Dyrk1a is the triplicated gene that causes decreased nuclear Cyclin D1 levels and early cortical neurogenic defects in a mouse model of DS [16]. Together, these studies emphasise the importance of DYRK1A gene dosage and activity. As a result of their co-translational activation loop phosphorylation the DYRKs are active once translated, however, there is growing evidence that some DYRKs are subject to additional post-translational modification and regulation. Autophosphorylation of DYRK1A allows binding of 14-3-3, which promotes DYRK1A catalytic activity [17,18]. In C. elegans, the DYRK homologue, minibrain kinase-2, is activated during oocyte maturation by cyclin-dependent kinase-1 (CDK1)-dependent phosphorylation of serine 68, a residue outside of the kinase domain that is required for full activity in vivo [19]. DYRK2 is phosphorylated at T33 and S369 (numbering corresponding to the short form of DYRK2) by the Ataxia telangiectasia mutated kinase (ATM) in response to genotoxic stress; this inhibits the ubiquitination of DYRK2, which can then phosphorylate S46 of the tumour suppressor p53 [20]. Finally, mass spectrometry of human DYRK4 expressed in HEK293 cells has identified phosphorylated Ser and Thr residues, indicating that DYRK4 is phosphorylated by other cellular protein kinases [21].
DYRK1B is implicated in promoting differentiation in several models: for example, it is expressed de novo during myogenesis [22] and undergoes differential splicing during adipogenesis [23]. Indeed, DYRK1B can promote cell cycle arrest by multiple mechanisms including promoting cyclin D1 (CCND1) degradation [24,25] by direct phosphorylation at T286 [25] and increasing the expression of the cyclin-dependent kinase inhibitors p21 CIP1 and p27 KIP1 [24,25]. Mutations in DYRK1B have been reported in an inherited form of metabolic syndrome associated with early-onset coronary artery disease, obesity, hypertension and diabetes [26]. In addition, DYRK1B is amplified [27,28] and mutated [29] in certain cancers and has been reported to promote cell survival [30][31][32]. Despite this, less is known about the post-translational regulation of DYRK1B. It has been suggested that oncogenic KRAS stimulates DYRK1B kinase activity [33] and that DYRK1B is a downstream effector of KRAS [34] but the molecular details of this regulation remain unclear.
There is growing interest in inhibiting DYRKs and several small molecule inhibitors of the class I DYRKs have been described including DYRK1B-selective inhibitors such as AZ191 [25] and dual 1A/1B inhibitors such as INDY [35] and Harmine [36]. These inhibitors are selective for the Ser/Thr kinase activity of the mature DYRKs [25,36], only inhibiting the Tyr kinase activity at very high doses, and are, therefore, useful in helping to define DYRK substrates and DYRK functions. To progress our interest in DYRK1B, we sought to identify DYRK1B autophosphorylation sites that were dependent on the Ser/Thr kinase activity of mature DYRK1B, since these might serve as biomarkers for DYRK1B activity and DYRK1B inhibitors. Here, we identify serine-421 (S421) as a site of DYRK1B trans-autophosphorylation that is inhibited by DYRK1B Ser/Thr kinase inhibitors and contributes to DYRK1B kinase activity. Remarkably, S421 is also phosphorylated in a MEK1/2-ERK1/2-dependent fashion in cells and by ERK2 in vitro, defining DYRK1B as a new substrate of ERK2 and providing a link between between two kinases involved in cell fate decisions. Finally, we show that mutations in DYRK1B that are found in cancer or metabolic syndrome either inhibit or are without effect on DYRK1B kinase activity. These results provide new insights into the regulation and role of DYRK1B.

Plasmids
For the mass spectrometry experiments, DYRK1B sequence (Swissprot Q9Y463; Embl AF205861) was ordered from Geneart with a c-terminal myc-tag and subcloned into pcDNA3. pcDNA3-DYRK1B was mutated by site-directed mutagenesis to generate catalytically inactive Y271F/Y273F and K140R constructs. For other experiments DYRK1B was amplified by PCR from pOTB7-DYRK1B (from Mammalian Gene Collection) and subcloned into pCMV-Tag2B and pEGFP-C3. pCMV-Tag2B-DYRK1B was mutated by site-directed mutagenesis to generate to catalytically inactive DYRK1B (D239A). pCGN-KRAS12V was from Adrienne Cox, Department of Pharmacology, University of North Carolina at Chapel Hill. The sequences of all oligos used are available upon request.

Analysis of in vitro autophosphorylation of recombinant DYRK1B by mass spectrometry
Recombinant DYRK1B expressed in insect cells (Carna Biosciences) was allowed to autophosphorylate in the presence of ATP. The protein was then resolved on a Novex gel followed by Coomassie staining. The Coomassie bands were reduced with DTT (Sigma), alkylated with iodoacetamide (Sigma), followed by digestion with: trypsin only; AspN and trypsin; chymotrypsin only; chymotrypsin and trypsin (all enzymes supplied by Roche). The resultant peptides were extracted and concentrated. The sample was chromatographed using the Dionex U3000 nanoflow chromatography system (ThermoFisher), the outlet flow ran directly into the Qstar Elite mass spectrometer (ABSciex) for analysis via the nanoflow probe at a flow rate of 300 nl/min. A 40 min reversed phase gradient was run using a 300 lm i.d. 9 5 mm C18 PepMap (Dionex ThermoFisher) trapping pre-column and a 75 lm i.d. 9 15 cm C18 ACE analytical column (Hichrom). The Qstar collected data in positive ion mode and an auto-switching setup was initiated with automatic precursor selection based on peak intensity and charge state. The subsequent data files generated were searched against the Swissprot database, using the Mascot Daemon software. The searches were then manually verified to confirm phosphorylation sites. From the MSMS data of the phosphorylated and nonphosphorylated peptides, the parent mass and key daughter ion masses were used to generate an MRM (multiple reaction monitoring) method on the 4000 Qtrap mass spectrometer (ABSciex).

Analysis of DYRK1B autophosphorylation in recombinant enzyme and cells by mass spectrometry using MRM
Recombinant DYRK1B enzyme was incubated with ATP in the presence or absence of 10 lM AZ'294 (an early DYRK1B HTS hit, with an IC 50 of \ 0.03 lM in an in vitro enzyme assay), and run on a Novex gel followed by Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity… 885 Coomassie Staining. Reduction, alkylation and digestion with trypsin was carried out as above. Wild-type or mutant myc-DYRK1B proteins were transiently expressed in COS-1 cells. DYRK1B proteins were enriched from whole cell lysates using anti-myc antibody, after treatment with 10 lM AZ'294 or 0.1 % DMSO for 2 h. Immuno-complexes were digested with trypsin prior to nano-liquid chromatography mass spectrometry. MRM was performed on the 4000 Qtrap mass spectrometer using the previously generated method from the recombinant analysis. The sample was chromatographed using the Dionex U3000 nanoflow chromatography system (ThermoFisher) the outlet flow ran directly into the 4000 Qtrap for analysis via the nanoflow probe at a flow rate of 700 nl/min. A 5 min reversed phase gradient was run using a 300 lm i.d. 9 5 mm C18 PepMap (Dionex ThermoFisher) trapping precolumn and a 75 lm i.d. 9 15 cm C18 ACE analytical column. The column was then equilibrated culminating in a 12.5 min run time per sample. The non-phosphorylated and phosphorylated masses of the pS421 containing tryptic peptide were monitored. Peak quantification was established using ABSciex software, this was manually verified and exported to Excel for further data analysis.

Preparation of cell extracts and Western blotting
Cells were lysed in ice-cold TG lysis buffer, assayed for protein content and fractionated by SDS-PAGE as described previously [38]. SDS-PAGE gels were transferred to Immobilon P membranes (Millipore), which were blocked in 0.1 % (v/v) Tween-20-TBS (tris-buffered saline) containing 5 % (w/v) powdered milk and probed with the indicated antibodies. Immune complexes were visualised with the ECL system (GE Healthcare).
In vitro DYRK kinase assay FLAG-DYRK1B [wild-type (WT), kinase-dead (KD, D239A) or S421A (SA)] were immunoprecipitated from whole cell lysates with anti-FLAG antibodies and then assayed for kinase activity by incubating with 50 lM Woodtide with two additional lysines attached to the N terminus to allow it to bind to P81 paper (KKISGRL-SPIMTEQ), 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM MgCl 2 , 0.1 mM [c-32 P]ATP in a total volume of 50 ll for 20 min at 30°C as described previously [7]. For each experiment, a single IP was used to generate three technical replicates of 32   The transfection mixes were incubated at room temperature for 20 min to allow the formation of siRNA complexes. Cells were seeded the previous day in Pen/ Strep-free medium to achieve a confluency of 50 % on the day of transfection. 500 ll of the transfection solution was added to each well of the tissue culture plate and the cells were incubated 37°C in a humidified incubator with 5 % (v/v) CO2 for 16 h. After this 16 h, the medium was changed to fresh Pen/Strep-free media containing DMSO or 1 lM Selumetinib. The cells were incubated for a further 48 h at 37°C in a humidified incubator with 5 % (v/v) CO2 prior to harvesting.

Analysis of cell cycle distribution and cell death
Cell cycle distribution (G 1 , S, G 2 /M) and the fraction of dead cells (those with sub-G1 DNA content) was assessed by staining fixed cells with propidium iodide followed by flow cytometry as described previously [38,39].

Results
Identification of S421 as a novel DYRK1B autophosphorylation site that promotes kinase activity When expressed in cells catalytically inactive DYRK1B exhibits enhanced migration on SDS-PAGE gels compared to wild-type suggesting that DYRK1B normally undergoes autophosphorylation in vivo [25]. Activation of DYRK1B requires auto-phosphorylation in cis at tyrosine 273 (Y273) within the activation loop. However, once translation is complete mature DYRK1B phosphorylates substrates on Ser/Thr residues in trans. We wished to develop an assay to monitor the kinase activity of mature DYRK1B in cells, hoping that this might serve as a biomarker to support the development of DYRK1B inhibitors. To determine if DYRK1B could autophosphorylate on other residues, we analysed potential auto-phosphorylation sites on recombinant DYRK1B by proteolytic digestion and mass spectrometry (MS). Using a combination of endoproteases we achieved 85 % coverage of DYRK1B and identified twelve phosphorylation sites including p-Y63 and p-Y273 that have been previously reported (Supplementary Table 1). A targeted MS method was developed to quantify these phosphorylation sites in Cos1 cells expressing Myc-DYRK1B using multiple reaction monitoring (MRM). This analysis identified four phosphorylation sites in cells but only one, S421 in the peptide IpSPLGALQHGFFR, was inhibited when cells were incubated in the presence of AZ'294, an early stage precursor of the DYRK1B inhibitor AZ191 [25] (Table 1). Cellular p-S421 was almost completely abolished when catalytically inactive DYRK1B mutants were expressed in Cos1 cells, including the activation loop mutant DYRK1B Y271F/Y273F or the catalytic lysine mutant DYRK1B K140R (kinase-dead or KD); these results demonstrated that p-S421 in cells was strongly dependent on DYRK1B kinase activity (Fig. 1a).
To progress this analysis further a p-S421 phosphospecific antibody was raised in rabbits and validated by its ability to detect wild-type DYRK1B but not the nonphosphorylatable DYRK1B S421A mutant when they were both expressed in HEK293 cells (Fig. 1b). Treatment of HEK293 cells with either of two DYRK1B inhibitors, AZ191 [25] or harmine [36], strongly inhibited p-S421 demonstrating that DYRK1B kinase activity was required for efficient S421 phosphorylation in cells (Fig. 1c). In contrast, we have previously shown that these inhibitors do not inhibit auto-phosphorylation at tyrosine, representing p-Y273 and p-Y63 [25], demonstrating that at the doses used they selectively inhibited the Ser/Thr kinase activity of mature DYRK1B. Since S421 is located in the DYRK1B kinase domain we determined if S421 phosphorylation had any role in DYRK1B kinase activity by comparing wild-type and DYRK1B S421A in kinase assays. DYRK1B proteins were expressed in HEK293 cells and isolated by immunoprecipitation prior to being assayed for kinase activity in vitro; kinase activity was normalised to the amount of wild-type or mutant DYRK1B that was recovered in the immunoprecipitation. These assays revealed that DYRK1B S421A exhibited a significant 50 % reduction in intrinsic kinase activity compared to wild-type DYRK1B (Fig. 1d) whereas kinase-dead DYRK1B D239A (KD) was completely defective. However, since only *50 % of DYRK1B was The resultant immuno-complexes were divided into two aliquots. The first aliquot was assayed for kinase activity against the synthetic substrate peptide Woodtide (50 lM) (upper panel). The second aliquot was separated by SDS-PAGE gels and transferred onto PVDF membrane. The amount of DYRK1B protein in each sample was determined by quantitative analysis of the immunoblot using Licor Odyssey (lower panel). DYRK1B kinase activity levels were normalised to the amount of DYRK1B present in each immunoprecipitate. Data were averaged from five separate experiments each with three technical replicates with each bar representing the mean ± standard deviation. Statistical analysis was performed using a two-tailed paired t test phosphorylated at S421 when over-expressed (Fig. 1a), this is probably an underestimate of the contribution of S421 to DYRK1B kinase activity. Thus, S421 phosphorylation contributes to DYRK1B kinase activity.

DYRK1B autophosphorylation at S421 occurs in trans
Based on the current model of DYRK activation [4,7], DYRK1B autophosphorylation of Y273 is thought to occur in cis through an intramolecular mechanism. To investigate if S421 auto-phosphorylation also occurred in cis, we expressed discrete DYRK1B proteins of different sizes (using either EGFP-DYRK1B or FLAG-DYRK1B) so that we could distinguish S421 phosphorylation in cis or in trans (Fig. 2a). FLAG-DYRK1B D239A (KD) expressed alone was not phosphorylated on S421, nor was wild-type EGFP-DYRK1B able to phosphorylate FLAG-DYRK1B S421A (SA). However, both EGFP-and FLAG-tagged wild-type DYRK1B auto-phosphorylated on S421, and this was blocked by AZ191 or harmine. Finally, FLAG-DYRK1B D239A (KD) was phosphorylated on S421 when co-expressed with wild-type EGFP-DYRK1B and this was blocked by AZ191 or harmine, suggesting this was an intermolecular phosphorylation (Fig. 2a). In addition, we expressed two different kinase-dead DYRK1B mutants-DYRK1B K140M or DYRK1B D239A -in HEK293 cells, immunopurified them and then incubated them in vitro with purified recombinant GST-DYRK1B; this is a far simpler system than co-expression in cells (Fig. 2a). These experiments demonstrated that purified GST-DYRK1B could indeed phosphorylate the two different kinase-dead DYRK1B mutants in vitro (Fig. 2b) and in both cases this in vitro trans-autophosphorylation was abolished by AZ191. Whilst we cannot rule out the potential involvement of an accessory protein co-purifying with the DYRK1B mutants, these results strongly suggest that one DYRK1B molecule can directly phosphorylate another on S421 in vitro. Recombinant GST-DYRK1B was also able to autophosphorylate at S421 under these conditions and this was again inhibited by AZ191. In summary, these data strongly suggest that S421 is a novel site of DYRK1B auto-phosphorylation that can proceed through an in trans mechanism.
Activation of the RAF-MEK-ERK1/2 signalling pathway also promotes S421 phosphorylation in cells In the course of this work we noted that whilst the DYRK1B S421A mutant was completely defective for p-S421 in cells (Fig. 1b), ATP-competitive inhibitors of DYRK1B Ser/Thr kinase activity left some residual p-S421 signal (Fig. 1c), raising the possibility that other cellular protein kinases might contribute to S421 phosphorylation. To investigate this we co-expressed DYRK1B together with an activated KRAS G12V mutant because: (i) KRAS drives activation of multiple protein kinase cascades and (ii) expression of activated KRAS has previously been proposed to activate DYRK1B by up to 20-fold [33]. Wildtype DYRK1B or kinase-dead DYRK1B D239A (KD) was transiently expressed with KRAS G12V for 24 h in HEK293 cells. DYRK1B proteins were then isolated from whole cell extracts by immunoprecipitation and assayed in an in vitro DYRK1B kinase assay; activity was again normalised to the amount of DYRK1B that was recovered in the immunoprecipitation (Fig. 3a). DYRK1B kinase activity was increased by co-expression with KRAS G12V ; however, this effect was modest and not statistically significant, reflecting that fact DYRK1B was already very active when expressed in HEK293 cells. Consistent with this, KRAS expression also failed to significantly increase S421 phosphorylation over the already high basal level (Fig. 3b). As a control, DYRK1B D239A (KD) exhibited negligible kinase Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity… 889 activity that was unaffected by KRAS G12V expression. Control blots confirmed that KRAS G12V was expressed and active since it activated endogenous ERK1/2 (Fig. 3b).
During these experiments, we observed that kinase-dead DYRK1B D239A (KD), which cannot auto-phosphorylate on S421 (Fig. 1c), became phosphorylated on S421 when coexpressed with KRAS G12V (Fig. 3b). This suggested that S421 could also be phosphorylated by a kinase other than DYRK1B in cells and that this kinase was activated by KRAS G12V . KRAS activates multiple effector pathways including the RAF-MEK1/2-ERK1/2 pathway and various PI3K-dependent pathways. However, we noted that S421 is immediately followed by a proline residue (P422) in a Ser-Pro motif, the minimum required for phosphorylation by proline-directed kinases such as ERK1/2. To address if activation of the ERK1/2 pathway alone could increase p-S421, DYRK1B D239A (KD) was expressed in HR1 cells [39]; these cells are derived from HEK293 cells and harbour the DCRAF:ER* fusion protein (Fig. 4a) that is activated by 4-hydroxytamoxifen (4HT). Upon activation of DCRA-F:ER*, DYRK1B D239A (KD) became phosphorylated on S421 and this was blocked by the highly selective MEK1/2 inhibitor, selumetinib (AZD6244/ARRY-142886) [40] (Fig. 4b). To investigate if S421 phosphorylation following RAF activation also occurred on wild-type DYRK1B, we used HD1B cells, a cell line that harbours DCRAF:ER* and exhibits tetracycline-inducible expression of DYRK1B [25]. When DYRK1B expression was induced with tetracycline, the DYRK1B protein displayed autophosphorylation on S421 (Fig. 4c). Treatment of these cells with the DYRK1B inhibitor AZ191 blocked p-S421 autophosphorylation but S421 phosphorylation was restored by DCRAF:ER* activation and this was again blocked by selumetinib. Thus, activation of the ERK1/2 signalling pathway was sufficient to drive p-S421 of DYRK1B in cells.

ERK2 directly phosphorylates DYRK1B on S421 in cells and in vitro
If DYRK1B is a direct substrate of ERK1/2 then the kinetics of S421 phosphorylation on DYRK1B should closely follow the kinetics of ERK1/2 activation in cells and should also match the phosphorylation of bona fide ERK1/2 substrates such as BIM EL [42] and RSK [43]. To address this we again expressed DYRK1B D239A (KD) in HR1 cells and stimulated them with 4HT for various times Wild-type or kinase-dead DYRK1B were transiently expressed together with empty vector or KRAS G12V for 24 h in HEK293 cells. a FLAG-DYRK1B proteins were isolated from whole cell extracts by immunoprecipitation and immuno-complexes were divided into two aliquots. The first aliquot was separated by SDS-PAGE, transferred onto PVDF membrane and immunoblotted for DYRK1B. DYRK1B levels were quantified on Licor Odyssey (upper panel). Data are from a single experiment representative of five separate experiments. The second aliquot was incubated with the synthetic DYRK substrate peptide Woodtide in an in vitro kinase assay to determine kinase activity. In vitro kinase activity was normalised to the amount of DYRK1B proteins present on the immuno-blot (lower panel). Data in the graph are presented as mean ± standard deviation from five separate experiments each with three technical replicates. Statistical analysis was performed using a two-tailed paired t test. b Whole cell extracts were fractionated by SDS-PAGE, transferred onto PVDF membrane and immuno-blotted with the indicated antibodies. Data are from a single experiment representative of five separate experiments with similar results to activate DCRAF:ER*. Activation of ERK1/2 was observed as early as 15 min after 4HT addition and persisted for the full 120 min timecourse (Fig. 5a). Increases in p-S421 DYRK1B tracked precisely with p-ERK1/2, being apparent from 15 min onwards; furthermore, inclusion of selumetinib at each timepoint abolished ERK1/2 activation and the p-S421-DYRK1B signal (Fig. 5a). In addition, phosphorylation of S421 on DYRK1B precisely matched the MEK1/2-dependent phosphorylation of RSK and BIM EL (monitored by it's characteristic reduction in mobility on SDS-PAGE) following activation of DCRAF:ER*. To determine if ERK2 could directly phosphorylate DYRK1B at S421, recombinant, active GST-ERK2 was incubated with FLAG-DYRK1B in an in vitro kinase assay (Fig. 5b). In this assay, wild-type DYRK1B was able to auto-phosphorylate on S421 in the absence of AZ191 (Lane 2) as shown by 32 P incorporation and pS421-DYRK1B western blot. However, 32 P was still incorporated into DYRK1B S421A in the absence of AZ191 (Lane 6), albeit to a lesser extent than wild-type DYRK1B, suggesting that there are other sites of DYRK1B auto-phosphorylation in vitro in addition to S421, consistent with our MS analysis (Table 1, Supplementary Table 1). In the presence of AZ191, DYRK1B auto-phosphorylation was blocked (Lanes 3 and 7). When active ERK2 was added to DYRK1B in the presence of AZ191, DYRK1B became phosphorylated on S421 (Lane 4) as shown by 32 P incorporation and p-S421 western blot and this was blocked by the ERK1/2 inhibitor SCH772984 [44] (Lane 5) demonstrating that S421-DYRK1B was a direct substrate of ERK2 in vitro. Interestingly, ERK2 was also able to phosphorylate DYRK1B S421A (Lane 8) although to a lesser degree, indicating that ERK2 may also be able to phosphorylate other sites on DYRK1B in vitro. Taken together these results demonstrated that in addition to auto-phosphorylation, DYRK1B is also phosphorylated on S421 by ERK1/2 in cells and in vitro.

Inhibition of MEK1/2-ERK1/2 signalling by selumetinib promotes DYRK1B expression
It has previously been show that first generation, pan-MEK1/2/5 inhibitors PD98059 and U0126 can increase DYRK1B mRNA and protein [22,45]. Notwithstanding the off-target effects of these drugs these results suggest that ERK1/2 signalling may normally repress DYRK1B expression. Our demonstration that ERK1/2 phosphorylates DYRK1B at S421 prompted us to examine DYRK1B expression using the more selective, second generation MEK1/2 inhibitor selumetinib [40]. Indeed, when we treated the melanoma cell line MelJuso (HRAS G13D / NRAS Q61L ) with 1 lM selumetinib this strongly and immediately inhibited ERK1/2 signalling and increased the expression of DYRK1B after a delay of 8 h. This was accompanied by an increase in DYRK1B mRNA which was apparent after 5 h of selumetinib treatment (Supplemental Fig. 1a). Treatment with actinomycin D, an inhibitor of transcription, prevented the increase in DYRK1B expression observed in response to selumetinib treatment (Supplemental Fig. 1b) suggesting that the increase in DYRK1B expression observed upon MEK inhibition reflects de novo gene transcription. Finally, similar results were obtained in the A375 melanoma cell line (BRAF V600E ) (Supplemental Fig. 1c), where selumetinib caused a dose-dependent inhibition of ERK1/2 signalling and an increase in expression of DYRK1B as well as BIM EL , which is known to be repressed by the ERK1/2 pathway [46]. Since inhibition of ERK1/2 Empty vector (EV), FLAG-DYRK1B wild-type or FLAG-DYRK1B S421A were transiently expressed in HEK293 cells. 4 h post-transfection, cells were treated with 10 lM AZ191 for a further 24 h to minimise DYRK1B autophosphorylation on S421. FLAG-DYRK1B proteins were then isolated from whole cell extracts by anti-FLAG immunoprecipitation and the resulting immuno-complexes were washed and used as a substrate in an ERK2 in vitro kinase assay in the presence of [c-32 P]ATP as described in ''Materials and methods''. Some of the assay tubes included the ERK1/2 inhibitor SCH772984. The terminated assay reactions were separated by SDS-PAGE, transferred onto PVDF membrane and 32 P incorporation was detected by autorad. Subsequently, the membrane was blotted with the indicated antibodies. Data are from a single experiment representative of two separate experiments with similar results signalling by selumetinib typically causes a G1 cell cycle arrest [47] (Fig. 6b), the increase in DYRK1B expression could simply be a consequence of cell cycle arrest rather than a specific consequence of reduced ERK1/2 signalling. To address this, we compared the effect of selumetinib with that of palbociclib (PD-0332991), a potent and highly selective inhibitor of CDK4 and CDK6 [48]. Whilst both drugs elicited a strong G1 cell cycle arrest (Fig. 6b) and inhibited phosphorylation of RB (Fig. 6c), only selumetinib inhibited ERK1/2 signalling and increased DYRK1B expression (Fig. 6c), demonstrating that the increase in DYRK1B was not simply a consequence of cell cycle arrest. Thus ERK1/2 signalling provides opposing regulatory inputs into DYRK1B: ERK1/2 can directly phosphorylate DYRK1B at S421, a residue that promotes kinase activity, but ERK1/2 signalling also represses DYRK1B expression.
DYRK1B has been proposed to function as a pro-survival protein kinase [30][31][32]. Since ERK1/2 inhibition by selumetinib typically results in a G1 cell cycle arrest with little cell death [47] we considered the possibility that DYRK1B expression might provide a survival signal following MEK1/2-ERK1/2 inhibition, thereby preventing cell death; that is, combined inhibition of MEK1/2 and DYRK1B would be synthetic lethal. To test this we treated MelJuso cells with selumetinib alone or in combination with the DYRK1B inhibitor AZ191 or in combination with DYRK1B siRNA, with GFP siRNA as a control (Fig. 7a). AZ191 treatment alone had no effect on cell death as judged by PARP cleavage (Fig. 7a) and the fraction cells with hypodiploid (sub-G1) DNA (Fig. 7b). Selumetinib treatment caused only a small increase in PARP cleavage and cell death. The combination of AZ191 and selumetinib caused greater cell death than either drug alone; however, whilst this was statistically significiant, it was only a very modest effect. We also observed a modest increase in cell death when we combined selumetinib with DYRK1B siRNA; however, this was not statistically significant (Fig. 7c). Finally, despite the induction of DYRK1B, we failed to observe any increase in cell death when selumetinib was combined with AZ191 or DYRK1B siRNA in A375 cells (data not shown). Thus, any synthetic lethal relationship between DYRK1B and MEK1/2 was at best weak and variable between cell lines.
Mutants of DYRK1B found in cancer or metabolic syndrome exhibit normal or reduced kinase activity Systematic sequencing of cancer genomes has revealed rare mutations in DYRK1B including L28P, R102H, S234G and Q275R [29]. However, the low incidence of these mutations calls into question whether they are 'drivers' or 'passengers' and no further functional information has been forthcoming. In addition, mutations in DYRK1B have been found in familial cases of metabolic syndrome [26,49] and include: gain-of-function mutations R102C and H90P; putative loss-of-function mutations, such as L28P, and apparently benign mutations, such as G352A and P578S. However, the impact of these disease-associated mutations on intrinsic kinase activity has not been described. To address this directly we engineered some of these mutations into individual DYRK1B constructs, expressed them in HEK293 cells, isolated them by immunoprecipitation and assayed their kinase activity in vitro. Our results revealed a consistent pattern: DYRK1B L28P (found in rare cases of metabolic syndrome as well as cancer) exhibited identical kinase activity to wild-type DYRK1B; DYRK1B R102H and DYRK1B S234G exhibited 40-50 % reduction in kinase activity and DYRK1B Q275R exhibited a 90 % reduction in kinase activity (Fig. 8a). We also directly compared the activity of the R102H mutant (found in cancer) and the R102C mutant (found in metabolic syndrome); both mutants exhibited a 50-60 % reduction in kinase activity (Fig. 8a). Finally, the relative activity of these DYRK1B mutants correlated well with their degree of phosphorylation at S421 (Fig. 8b) suggesting that in the absence of strong inputs from the ERK1/2 pathway p-S421 correlated well with intrinsic activity. Thus mutations in DYRK1B that are found in cancer (L28P, R102H, S234G and Q275R) or metabolic syndrome (L28P, R102C) have either no effect on intrinsic kinase activity or reduce kinase activity, in one case (DYRK1B Q275R ) severely.

Discussion
Trans-autophosphorylation at S421 contributes to DYRK1B activity Our results identified S421 as a site of DYRK1B autophosphorylation since it was present in purified recombinant wild-type DYRK1B incubated in vitro with ATP, absent in kinase-dead DYRK1B and strongly (though not completely) inhibited by DYRK1B inhibitors in vitro or in cells. These drugs do not prevent autophosphorylation on tyrosine (largely Y273, but also Y63) in cells [25] except at high concentrations [36], consistent with phosphorylation of Y273 and S421 proceeding through different mechanisms. Indeed, we have now shown that S421 autophosphorylation can proceed through an intermolecular trans-phosphorylation mechanism whereas Y273 is an intramolecular cis-phosphorylation event. To our knowledge, this is the first description of a serine or threonine auto-phosphorylation site on DYRK1B. S520 in DYRK1A was previously identified as a site of auto-phosphorylation that was required for full DYRK1A kinase activity [18]; in this case S520 auto-phosphorylation increased 14-3-3b binding to DYRK1A. However, phosphorylation of S520 in DYRK1A took place in cis, in contrast to the intermolecular phosphorylation of S421 in DYRK1B described here. Indeed, given the position of S421, situated away from the kinase active site, it is difficult to imagine it could be phosphorylated in cis through an intramolecular mechanism. Regardless, it seems that S421 phosphorylation also contributes to DYRK1B kinase activity, either directly or by an indirect mechanism involving accessory proteins, since the S421A mutant exhibited a consistent 50 % reduction in kinase activity; indeed, this is likely to be an underestimate of the impact of the S421A mutant since only half of over-expressed DYRK1B was phosphorylated at S421. Whilst a twofold modulation of kinase activity may seem modest, it is important to remember that even a 50 % increase in expression and activity of the closely related DYRK1A has profound physiological effects in mice and phenocopies some Down's syndrome defects [11,  15] whilst a 50 % decrease in DYRK1A causes even more dramatic effects in Drosophila, mice and human patients including microcephaly, growth retardation and behavioural defects [11,50,51]. S421 occurs close to the highly conserved arginine residue (AARIS 421 PL) shortly after alpha helix H/kinase sub-domain XI [3,10]. Serine and threonine are the first and third most common residues at this position across the kinome as a whole, whilst the Cancer Genome Atlas (TCGA) reveals that the position equivalent to S421 is ranked 25 th in terms of frequency out of the 2500 identified mutation positions across kinase domains, suggesting a potential wider significance for phosphorylation at this site. Of the 61 human CMGC kinases listed at http://kinase.com/human/kinome/groups/cmgc.aln only 7 lack a Ser or Thr at the position corresponding to Ser421 of DYRK1B. More significantly, S421 in DYRK1B is a Pro-directed site (Ser-Pro) and within the closely related human DYRK family (DYRK1A, 1B, 2, 3 & 4 and the four HIPKs) the corresponding site is a Ser-Pro or Thr-Pro in all cases except DYRK1A (Gln-Pro) raising the possibility that these kinases may also be regulated by phosphorylation at this site. This site is also Ser-Pro or Thr-Pro in DYRKs from lower eukaryotes including S. pombe Pom1p, S. cerevisiae Yak1p, C. elegans MBK-1 and MBK-2 and D. melanogaster dDYRK2 and dDYRK3, indicating evolutionary conservation of the site. In terms of functional relevance, intermolecular phosphorylation at S421 suggests that DYRK1B may be able to form dimers. Indeed, the structure of the closely related DYRK1A [10] revealed multiple copies per unit cell with Q469 of DYRK1A, corresponding to S421 of DYRK1B, well away from the ATP binding pocket at the interface between DYRK1A dimers. It is currently unclear if DYRK1B is a dimer in cells or whether it is more or less active as a dimer or monomer but it will be interesting to determine if S421 phosphorylation influences dimerisation. Alternatively, S421 phosphorylation may influence substrate interactions, which would be consistent with the reduction in kinase activity that we observe for the S421A mutant. For example, the closely related DYRK4 exhibits an alternative splicing donor site in exon 19 which removes three amino acids (DCLV) at the end of the a-helix H, immediately prior to the conserved Arg in subdomain XI [21]. This region is thought to contribute to the substrate-binding structure [52] and it is notable that DYRK4 DCLV exhibits reduced kinase activity against exogenous substrates compared to the full length DYRK4 [21]. Thus, it is possible that changes to this region in DYRK1B, such as phosphorylation of S421, might contribute to ordering of the substrate-binding structure. Such speculation will need to be addressed by future structural studies.

ERK1/2 can phosphorylate DYRK1B at S421
We sought to identify DYRK1B autophosphorylation sites as biomarkers for DYRK1B kinase activity and identified S421 as a phosphorylation site that promotes DYRK1B activity. However, we found that S421 is also phosphorylated by ERK1/2; whilst this disqualifies S421 as a unique DYRK1B activity biomarker it reveals a new mode of cross talk between ERK1/2 and DYRK1B.
We noted that inhibitors of DYRK1B Ser/Thr kinase activity left a residual p-S421 signal in cells, prompting a search for other kinases that might target this site. For this purpose we co-expressed DYRK1B with KRAS G12V , since KRAS has several effectors (PI3K, RAL, etc.,) and activates multiple protein kinase signalling pathways. In contrast to previous reports of a 9-to 20-fold activation [33] we consistently found that KRAS G12V caused only a very modest (10-15 %) increase in activity that was not statistically significant. This was not due to a deficit in our KRAS construct as KRAS G12V strongly activated endogenous ERK1/2 in the same transfections. These results suggest that any effect of activated KRAS on DYRK1B effector pathways such as Hedgehog [34] may be independent of DYRK1B intrinsic kinase activity, though we cannot rule out a possible effect on DYRK1B substrate binding (see above) that might be missed in an assay that employs simplified peptide substrates. However, these experiments also revealed that KRAS G12V could actually increase DYRK1B phosphorylation at S421. Through a combination of conditional kinases and selective kinase inhibitors we were able to show that ERK1/2 activation alone could increase p-S421, commensurate with established ERK1/2 substrates, and ERK2 could phosphorylate S421 in vitro. This is the first report to clearly define DYRK1B as a substrate of ERK2. A previous study suggested that ERK2, JNK or p38 might phosphorylate DYRK1B but stopped short of specifying the kinase, identifying the phosphorylation site or the effect on DYRK1B function [53].
Together with previous studies, our results reveal a complex interplay between ERK1/2 signalling and DYRK1B. Since phosphorylation of S421 may enhance DYRK1B kinase activity, ERK1/2 inhibition could decrease DYRK1B activity. In contrast, previous studies have indicated that inhibition of ERK1/2 signalling may increase DYRK1B expression [22,45] and we also observed this with the highly selective MEK1/2 inhibitor selumetinib, suggesting that ERK1/2 signalling can repress DYRK1B expression. Thus, ERK1/2 inhibition may reduce the activity of existing DYRK1B but also increase de novo DYRK1B expression, providing a mechanism for fine tuning DYRK1B activity. It is well known that the ERK1/2 pathway is a key regulator of the cell cycle, promoting proliferation or cell cycle arrest depending on the magnitude or duration of ERK1/2 activity [54]. DYRK1A and DYRK1B have also emerged as regulators of the cell cycle; both can promote the turnover of CCND1, increase expression of p21 CIP1 and/or p27 KIP1 and thereby promote cell cycle arrest and differentiation [22,24,25,45,55]. Indeed, even modest (*50 %) changes in DYRK1A expression and activity exert profound effects on the cell cycle [56]. ERK1/2-dependent regulation of DYRK1B adds another level of control, providing a link between two cell fate signalling pathways.
There is growing interest in identifying synthetic lethal drug combinations in the treatment of cancer [57]. For example, whilst selumetinib exerts a largely cytostatic effect, combining the BH3-mimetic ABT-263 with selumetinib causes up to 80 % cell death in colorectal cells and delays the onset of acquired resistance to selumetinib [47]. The increase in DYRK1B expression following MEK1/2-ERK1/2 inhibition prompted us to investigate whether inhibition of DYRK1B might similarly transform the cytostatic effects of selumetinib to promote cell death. Indeed, Gao et al. recently described a twofold increase in H292 cell death when the pan-MEK1/2/5 inhibitor U1026 was combined with DYRK1B siRNA [45], although they did not report the actual percentage of dead cells. We also observed a twofold increase in cell death in MelJuso cells when selumetinib was combined with DYRK1B siRNA or the AZ191 DYRK1B inhibitor. However, the percentage of dead cells observed under these conditions was modest, being less than 20 %, and was not observed in a second cell line (A375) that exhibited strong DYRK1B upregulation following ERK1/2 inhibition. Thus, DYRK1B and MEK1/ 2 do not seem to exhibit a robust synthetic lethal relationship that might form the basis for a rational drug combination.
DYRK1B mutants found in metabolic syndrome or cancer exhibit normal or reduced kinase activity DYRK1B is over-expressed due to gene amplification in certain tumours, including pancreatic cancer [27,28], and this has led to the suggestion that DYRK1B is a potential oncogene. In addition, cancer genome sequencing has revealed mutations in DYRK1B including L28P, R102H, S234G and Q275R [29]. Both L28 and R102 lie outside the DYRK1B kinase domain; L28 lies outside any known conserved regions whilst R102 is situated between the DYRK homology (DH) box and the kinase domain. S234 is conserved in the class 1 DYRKs and immediately precedes the catalytic loop in kinase subdomain VIb whilst Q275 is conserved in all DYRKs and follows the critical Y273 in the kinase activation loop within subdomain VIII. In addition, L28P and R102C mutations were found in cases of metabolic syndrome [26,49]. However, these diseaseassociated mutations were found at low frequency and their impact on DYRK1B activity was not assessed.
We found that these mutations were either neutral with respect to DYRK1B activity (L28P) or caused a consistent reduction in kinase activity (R102H, R102C S234G and Q275R); in the case of the Q275R mutation this effect was severe, with an approximate 90 % reduction in kinase activity. Q275 lies within the activation loop of DYRK1B; whilst activating, oncogenic mutations are found in this region at significant frequency in some protein kinases, mutations in this loop, including the introduction of bulky and highly positive side chain such as arginine, are also known to inhibit activity by impacting on activation loop conformation and flexibility. At this stage we cannot rule out the possibility that these mutations might confer kinase-independent properties upon DYRK1B; for example, by driving DYRK1B to a specific cellular location or engaging with particular partner proteins. Indeed, some of the effects of DYRK1B in metabolic syndrome were apparently independent of DYRK1B kinase activity [26]. Regardless, our results suggest that these DYRK1B mutations are unlikely to be gain-of-function in terms of intrinsic kinase activity and some may even be loss-offunction mutations. Since DYRK1B can promote cell cycle arrest and differentiation [22,24,25,45,55,56], such lossof-function mutations suggest that any oncogenic properties of DYRK1B are, at best, context-dependent; indeed, they may even suggest tumour suppressor functions for wild-type DYRK1B.
In summary, we demonstrate that phosphorylation of S421 contributes to DYRK1B kinase activity and that S421 is a site for DYRK1B autophosphorylation and phosphorylation by ERK1/2, defining DYRK1B as a new substrate of ERK1/2. Our results reveal opposing effects of ERK1/2 signalling on DYRK1B which may serve to fine tune DYRK1B activity. However, despite the ability of ERK1/2 to phosphorylate S421, KRAS G12V does not increase DYRK1B kinase activity. This may be because DYRK1B expressed in cells is already active so that any effects of KRAS are modest and incremental; this may require single cell analysis to resolve. Alternatively, different KRAS effectors may exert opposing effects on DYRK1B; so whilst activation of RAF-MEK-ERK may promote S421 phosphorylation, that would enhance activity, other KRAS effector pathways may exert opposing effects on DYRK1B. Indeed, our mass spectrometry analysis has identified at least two other phospho-Ser sites on DYRK1B expressed in cells that are not autophosphorylation sites; the kinases for these sites and the functional consequences remain unknown. Finally, mutants of DYRK1B found in cancer or metabolic syndrome are either neutral with respect to kinase activity or exhibit significant decreases in kinase activity. At least one of these, R102C in metabolic syndrome, is defined as a gain-of-function mutation [26] but clearly exhibits reduced kinase activity, raising the possibility that some effects of DYRK1B may not be strictly dependent on kinase activity.