Sprouty2-Mediated Inhibition of Fibroblast Growth Factor Signaling Is Modulated by the Protein Kinase DYRK1A

Raf-MEK-extracellular signal-regulated kinase (Erk) signalinginitiated by growth factor-engaged receptor tyrosine kinases(RTKs) is modulated by an intricate network of positive andnegative feedback loops which determine the specificity andspatiotemporal characteristics of the intracellular signal.Well-known antagonists of RTK signaling are the Sprouty proteins.The activity of Sprouty proteins is modulated by phosphorylation.However, little is known about the kinases responsible for theseposttranslational modifications. We identify DYRK1A as one ofthe protein kinases of Sprouty2. We show that DYRK1A interactswith and regulates the phosphorylation status of Sprouty2. Moreover,we identify Thr75 on Sprouty2 as a DYRK1A phosphorylation sitein vitro and in vivo. This site is functional, since its mutationenhanced the repressive function of Sprouty2 on fibroblast growthfactor (FGF)-induced Erk signaling. Further supporting the ideaof a functional interaction, DYRK1A and Sprouty2 are presentin protein complexes in mouse brain, where their expressionoverlaps in several structures. Moreover, both proteins copurifywith the synaptic plasma membrane fraction of a crude synaptosomalpreparation and colocalize in growth cones, pointing to a rolein nerve terminals. Our results suggest, therefore, that DYRK1Apositively regulates FGF-mitogen-activated protein kinase signalingby phosphorylation-dependent impairment of the inhibitory activityof Sprouty2.

INTRODUCTION

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INTRODUCTION

Signaling pathways initiated by receptor tyrosine kinases (RTKs)regulate fundamental cellular processes such as proliferation,differentiation, apoptosis, and cell migration (for a review,see reference 44). RTKs are activated by the binding of specificligands, which induces their dimerization and autophosphorylationof tyrosine residues in their cytoplasmic tails. These eventslead to the appearance of specific binding sites for a widevariety of proteins initiating intracellular signaling cascades(for a review, see reference 41). An essential pathway downstreamof RTK signaling is the mitogen-activated protein kinase (MAPK)cascade, which is composed of members of the Raf family, MAPKkinases (MEK), and extracellular signal-regulated kinase (Erk)kinases. The signal output from the Erk cascade is finely modulatedby positive and negative regulators that contribute to determiningboth the magnitude and the duration of Erk signaling (14). Animportant class of negative regulators of RTK-dependent pathwaysis the Sprouty (Spry) family of proteins. In Drosophila melanogaster,dSpry interferes with signaling by fibroblast growth factor(FGF) and epidermal growth factor (EGF) (8, 20). In mammals,four dSpry orthologs have been identified that function as negativeregulators of RTK signaling in vivo (for a recent review, seereference 37). Several modes of action have been proposed forSpry proteins, but none of them satisfactorily explains thediversity of their inhibitory activities. These range from actingas an uncoupling factor at the level of FRS2/GRB2/Sos (22, 32)or Cbl/RTK complex formation (16, 42) to interfering in Rasor Raf activation (19, 43, 49). In addition, Spry activity ismodulated at different levels by posttranslational modificationssuch as palmitoylation (28) and phosphorylation events in tyrosine,serine, and threonine residues (10, 16, 22, 32, 36).

Inappropriate spatial or temporal activation of MAPK cascadesupon growth factor stimulation is often implicated in tumorprogression and human developmental pathologies (for a recentreview, see reference 45). One of the most common human developmentaldisorders is caused by trisomy of chromosome 21. Among the humanchromosome 21 genes, DYRK1A has been proposed as a major contributorto Down syndrome (12, 15). The phenotypes associated with DYRK1Agene dosage imbalance in vivo are thought to be caused by alterationsof specific gene expression patterns and/or changes in intracellularsignaling outcomes (3, 6). DYRK1A encodes a protein kinase belongingto an evolutionarily conserved family of protein kinases knownas DYRKs (dual-specificity tyrosine [Y] phosphorylation-regulatedkinases). DYRK1A is highly expressed in the brain during developmentand also in adults, where it is detected at high levels in olfactorybulb, spinal cord, cerebellar cortex, and the majority of motornuclei (35). Loss of Dyrk1A function is lethal at embryonicmidstages, and these mice show severe developmental retardation(17). Heterozygotes are viable but present a reduced size atbirth that is maintained at adult stages. Size reduction ismost noticeable in organs such as liver and brain (17). Indeed,several brain structures are differently affected; for example,there is a reduction in the complexity of the neuronal dendritictree in the cortex (5) and also a reduction in the number ofneurons in the superior colliculus (17). These data, togetherwith the expression pattern of the protein, suggest a role forDYRK1A in the development and homeostasis of the central nervoussystem. At the molecular level, several substrates and proteinpartners for DYRK1A have been reported (for a recent review,see reference 13). The list includes both cytosolic and nuclearproteins, suggesting specific roles for DYRK1A in differentsubcellular compartments. Regarding DYRK1A actions outside thenucleus, DYRK1A overexpression has been shown to potentiatethe differentiation effects of nerve growth factor in the neuron-likecellular model PC12 (29). This effect, which is independentof kinase activity, correlates with increased levels of activatedErk1/2, a downstream effector of the neurogenic activity ofnerve growth factor.

Despite all this, there is still a lack of understanding ofthe DYRK1A role in intracellular signaling pathways. Here, weshow that the negative regulator of FGF signaling Spry2 is anovel DYRK1A partner and substrate. Based on our findings, wepropose that DYRK1A promotes FGF signaling by acting as a negativeregulator of RTK antagonistic Spry proteins.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Plasmids. Expression plasmids encoding the DYRK1A variants have been describedpreviously: hemagglutinin (HA)-tagged variants (pHA derivatives),variants N-terminally fused to glutathione S-transferase (GST)(pGST derivatives), and the DYRK1A fragment 590-611 fused tothe enhanced green fluorescent protein (GFP) (pGFP-Hrep) (1,2). The expression plasmid pHA-DYRK1A ${Delta}$ His was generated by site-directedmutagenesis. The expression plasmid for FGF receptor (FGFR;pMyc-FGFR1) (22) was kindly provided by E. Nishida (Departmentof Cell and Developmental Biology, Kyoto University, Japan).pSG.Gal-Elk (31) was a kind gift from P. E. Shaw (School ofBiomedical Sciences, University of Nottingham, United Kingdom).pE1bGal-luc was as described previously (11), and pRL-Null wasfrom Promega.

The Spry2 expression plasmid (pFlag-hSpry2) (33) was kindlyprovided by G. R. Guy (Institute of Molecular and Cell Biology,Singapore). To make plasmids pMyc-Spry2 and pGST-Spry2, theopen reading frame (ORF) of human Spry2 was amplified by PCRand subcloned into the EcoRI and XhoI sites of pCDNA3-Myc orpGEX-5X-3 (Amersham Biosciences), respectively. For generationof pGST-Spry2-(1-164) and pGST-Spry2-(1-255), AflII/XhoI andNcoI/XhoI fragments, respectively, were removed from pGST-Spry2,and the ends were filled with T4 DNA polymerase and religated.To generate pFlag-Spry2 ${Delta}$ 164-255, a DNA fragment encoding aminoacids 255 to 315 of human Spry2 was amplified by PCR and thensubcloned into the AflII/XhoI sites of pFlag-Spry2. Constructsfor Y55F, T75A, T122A, and T126A Spry2 mutants were generatedby site-directed mutagenesis. Plasmids pFlag-Spry1, pFlag-Spry3,and pFlag-Spry4 were generated by replacing a BamHI/XhoI fragmentcontaining the Spry2 ORF in pFlag-Spry2 with DNA fragments containingthe corresponding Spry ORFs. These fragments were obtained byPCR using IMAGE Consortium cDNA clones 6028920 (human Spry1),2208245 (human Spry3), and 6821944 (mouse Spry4) as templates.The clones were purchased from the RZPD German Resource Centerfor Genome Research. Nucleotide changes and small deletionswere made by oligonucleotide-directed mutagenesis using theQuikChange site-directed mutagenesis kit according to the manufacturer'sinstructions (Stratagene). All plasmids generated by PCR orsite-directed mutagenesis, as well as all the in-frame fusions,were verified by DNA sequencing.

Yeast two-hybrid screening. Full-length human DYRK1A cDNA (754-amino-acid isoform) was subclonedinto pAS2 (Clontech) and used as bait to screen a fetal humanbrain cDNA library cloned into pACT2 (Clontech). Positive clonesfor HIS3 selection were rescreened for LacZ expression. Plasmidscontaining the prey sequences were rescued and checked by back-transformationwith the bait. Library screening and β-galactosidase measurementswere done according to Clontech protocols.

Cell culture and transfection. HEK-293 and HEK-293T cell lines were maintained at 37°Cin Dulbecco's modified Eagle's medium, supplemented with 10%fetal calf serum (FCS) and antibiotics (100 units/ml penicillinand 100 µg/ml streptomycin). Transient transfections wereperformed using the calcium phosphate method, and cells wereprocessed 48 h after transfection. For preparation of dissociatedcortical neuron cultures, mouse embryos at embryonic day 15were obtained from anesthetized pregnant females. Cerebral corticeswere isolated and mechanically dissociated in phosphate-bufferedsaline (PBS) with 5 mg/ml glucose. Cells were plated onto glasscoverslips (80 cells/mm²) previously coated with 2 µg/mlpoly-L-lysine (Sigma). Cells were maintained in neurobasal medium(Gibco) supplemented with B27 (Gibco) and 1% inactivated horseserum (Gibco).

Western blotting. Samples were resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE), transferred onto Hybond C membranes(Amersham), and blocked with 10% skim milk in 10 mM Tris-HCl(pH 7.5)-100 mM NaCl (TBS) plus 0.1% Tween 20 (TBS-T). Membraneswere incubated with primary antibodies (in 5% skim milk in TBS-T)overnight at 4°C, except in the case of PY20 antibody, whenbovine serum albumin replaced skim milk for both blocking andantibody incubation. After being washed with TBS-T, membraneswere incubated for 45 min at room temperature with horseradishperoxidase-conjugated rabbit anti-mouse and goat anti-rabbitantibodies (Dako) (in 5% skim milk in TBS-T) and then washedagain with TBS-T. Detection was by enhanced chemiluminescencewith Supersignal West Pico (Pierce). Chemiluminescence was determinedwith a Fuji LAS-3000 image analyzer. Quantification of the datawas performed using Multi Gauge software (v3; Fuji PhotoFilm).The following antibodies were used as primary antibodies: mousemonoclonal antibody (MAb) anti-HA (Covance), MAb anti-Flag M2(Sigma), rabbit polyclonal antibody (rAb) anti-Myc (Santa Cruz),MAb anti-GFP JL-8 (BD Biosciences), rAb anti-lamin B1 (SantaCruz), MAb anti-SNAP-25 (BD Biosciences), MAb antisynaptophysin(Sigma), rAb anticalretinin (Swant), rAb anti-Erk2 (Santa Cruz),MAb anti-phospho-Erk1/2 (Thr202/Tyr204) (Cell Signaling), rAbanti-B-Raf (Santa Cruz), rAb anti-c-Cbl (Santa Cruz), rAb anti-GRB2(Santa Cruz), rAb anti-FGFR1 (Santa Cruz Biotech), rAb anti-DYRK1A(1), MAb anti-DYRK1A (Abnova), MAb anti-phospho-Tyr PY20 (BDBiosciences), and rAb anti-Spry2 (Upstate Biotechnology).

Mouse brain subfractionation. The fractionation procedures described here are summarized inFig. 6A. All procedures were done at 4°C. Equal volumesof every fraction were resuspended in 6x Laemmli buffer (LB)and analyzed by Western blotting. For density gradient sucrosefractionation, two mouse brains were dissected and homogenizedin 10 volumes of sucrose buffer (0.32 M sucrose, 10 mM HEPES[pH 7.4], 1 mM EGTA, 1.5 mM MgCl₂, protease inhibitor cocktail[PIC; Roche], and phosphatase inhibitors [PI; 10 mM sodium orthovanadate,30 mM sodium pyrophosphate]) with a glass-Teflon homogenizerusing 10 up-and-down strokes at 700 rpm. The homogenate (H)was centrifuged for 10 min at 800 x g, yielding a pellet (P1)containing nuclei and any unbroken cells and a supernatant (S1)free of nuclei. The supernatant was placed at the top of a 0.4to 2 M discontinuous sucrose gradient (nine 1-ml layers of 0.2M increments) and fractionated by centrifugation for 3 h at33,000 rpm in a SW41 rotor (Beckman). Aliquots of 0.2 ml wereextracted from top to bottom of the gradient.

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FIG. 6. DYRK1A and Spry2 associate with the plasma membranes of synaptic terminals. (A) Schematic representation of the subcellular fractionation procedures used for the analyses in panels B to D. Centrifugation steps are indicated with a semicircular arrow. Full details are provided in Materials and Methods. (B) Homogenates from mouse brain were obtained as described in Materials and Methods. Equal volumes of the homogenate (H), the nuclear pellet (P1), and the postnuclear fraction (S1) were analyzed by Western blotting with the indicated antibodies. (C) The postnuclear fraction (S1) from mouse brain homogenates was fractionated by centrifugation in a discontinuous sucrose gradient. Equal volumes of the first 18 alternate fractions were analyzed by Western blotting with specific antibodies, as indicated. (D) The postnuclear fraction (S1) from mouse brain homogenates was fractionated by differential centrifugation. Equal volumes of the fractions obtained were analyzed by Western blotting with specific antibodies, as indicated. P2, crude synaptosomal fraction; S2, cytosolic and microsomal fraction; P3, microsomal fraction; S3, soluble cytosolic fraction; LP1, synaptosomal plasmatic membrane fraction; LP2, crude synaptosomal vesicle fraction; LS2, synaptosolic fraction.

Fractionation by differential centrifugation was performed essentiallyas described previously (26). The postnuclear S1 fraction wascollected and centrifuged for 15 min at 9,200 x g. The supernatant(S2) was removed, and the pellet (P2) was washed by resuspensionin 9 ml of sucrose buffer and centrifuged for 15 min at 10,200x g, yielding a supernatant (S2') and a pellet (P2'). The supernatantsS2 and S2' were pooled and centrifuged for 1 h at 165,000 xg, yielding a pellet (P3) and soluble fraction (S3). The pelletP2' was resuspended in 1 ml of sucrose buffer, and 8 ml of coldwater (supplemented with PIC and PI) was added. The whole suspensionwas subjected to seven up-and-down strokes at 1,500 rpm witha glass-Teflon homogenizer. The resulting P2' lysate was rapidlytransferred to a tube (to which was added 64 µl of 1 MHEPES, pH 7.4), kept on ice for 30 min, and then centrifugedfor 20 min at 25,000 x g, yielding a lysate pellet (LP1) anda supernatant. The supernatant was collected and centrifugedfor 2 h at 165,000 x g, yielding a lysate pellet (LP2) and lysatesupernatant (LS2). All centrifugations were done with a TLA-100.3rotor (Beckman).

Immunohistochemistry and immunofluorescence. Animals were housed in the Animal Facility of the Parc de RecercaBiomedica de Barcelona and were sacrificed in accordance withthe recommendations and protocols for the proper use of laboratoryanimals approved by the local ethics committee. Mice were anesthetizedand transcardially perfused with cold PBS and 4% paraformaldehydein 0.1 M phosphate buffer, pH 7.4. Brains were removed, postfixedin the same fixative for 24 h, and then cryoprotected in 30%sucrose in PBS. For immunohistochemistry, free-floating sections(40 µm) were blocked in blocking buffer (BB; 0.25% gelatin,0.2% Triton X-100 in PBS) with 10% FCS and incubated overnightat 4°C with primary antibodies (rAb anti-DYRK1A or rAb anti-Spry2)diluted in BB with 1% FCS. For immunoperoxidase detection, sectionswere incubated with a goat anti-rabbit biotinylated secondaryantibody (1:200; Vector Labs) followed by ABC complex (VectorLabs) and reacted with 0.03% diaminobenzidine (Sigma) and 0.003%hydrogen peroxide. Finally, sections were dehydrated and mountedon slides. For immunofluorescence detection, 20-µm sectionson slides were blocked in BB with 10% FCS and then incubatedovernight at 4°C with primary antibodies (MAb anti-DYRK1Aand rAb anti-Spry2) diluted in BB with 1% FCS. Secondary antibodiesused were goat anti-rabbit antibody-fluorescein isothiocyanateconjugate (Southern Biotech) and goat anti-mouse antibody-TexasRed conjugate (Jackson ImmunoResearch). Samples were counterstainedwith 4',6'-diamidino-2-phenylindole (DAPI) to visualize cellnuclei and mounted in Vectashield (Vector Labs). Samples wereviewed under a Leica DMI 6000B microscope, and fluorescenceimages were acquired with a Leica DFC350 FX camera.

For immunolocalization studies in primary cortical neuron cultures,cells in coverslips were processed after 10 days in culture.Cells were fixed in 4% paraformaldehyde in PBS for 15 min atroom temperature, and the immunofluorescence assay was performedas described above. Goat anti-mouse antibody-Alexa 555 and goatanti-rabbit antibody-Alexa 488 (Molecular Probes) were usedas secondary antibodies. Samples were viewed under a Leica TSCSPE confocal microscope, and the images were captured in a consecutiveconfocal Z-sectioning of 0.37 µm (approximately six sections).The Z-stack projection image was generated using ImageJ processingsoftware (http://rsb.info.nih.gov/ij).

GST fusion protein expression in bacteria and pull-down assays. GST fusion expression constructs were transformed into Escherichiacoli BL21(DE3)pLysS. Protein expression was induced with 0.1mM isopropyl-β-D-thiogalactopyranoside for 3 h at 37°Cfor unfused GST, 4 to 6 h at 22°C for GST-Spry2 proteins,and 8 h at 20°C for GST-DYRK1A. Induced recombinant proteinswere bound to glutathione beads and, when required, were elutedwith 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8, anddialyzed against a buffer containing 50 mM HEPES (pH 7.4), 150mM NaCl, and 2 mM EDTA. Protein concentration was determinedwith a colorimetric assay (bicinchoninic acid protein assaykit; Pierce) and/or by Coomassie blue staining of SDS-polyacrylamidegels and comparison with a standard.

For pull-down assays, soluble cell lysates were incubated overnightat 4°C with 5 µg of unfused GST or GST-Spry2 (fulllength or mutants) immobilized on glutathione-Sepharose beadsthat had been previously equilibrated in lysis buffer (50 mMHEPES [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 [NP-40],PIC, and PI). After binding, beads were washed four times withlysis buffer, and the bound protein was eluted by boiling samplesfor 5 min in SDS buffer. Samples were resolved by SDS-PAGE,and proteins were detected by immunoblotting.

Immunoprecipitation. For soluble cell lysates, cells were washed twice in PBS andlysed in buffer A (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mMEDTA, 1% NP-40, 10% glycerol, PIC, and PI). Lysates were clarifiedby centrifugation and diluted with 2 volumes of buffer A withoutNP-40. Lysates were incubated for 6 h at 4°C with proteinG-Sepharose beads (Amersham) prebound with 5 µg of anti-HAor anti-Flag antibodies, and then beads were washed with lysisbuffer A.

For immunoprecipitation of endogenous DYRK1A protein from mousebrain, brains from 2- to 4-month-old mice were dissected andhomogenized mechanically with a Dounce homogenizer in 5 ml oflysis buffer B (10 mM HEPES [pH 7.4], 150 mM NaCl, 1 mM EDTA,0.1 mM MgCl₂, 0.2% Triton X-100, 10% glycerol, PIC, and PI).The homogenates were sonicated with 5 pulses of 5 s with anintensity of 0.5 in a Sonifier 250 (Branson) and clarified bycentrifugation for 10 min at 16,000 x g at 4°C. Ten milligramsof soluble lysates was precleared for 1 h at 4°C with equilibratedprotein A-Sepharose and incubated overnight at 4°C with10 µg of either rAb anti-DYRK1A or nonspecific rabbitimmunoglobulin G (IgG; Sigma). Then, the immunocomplexes wereincubated with protein A-Sepharose for 2 h at 4°C, and beadswere washed with lysis buffer B. Finally, both lysates and theimmunoprecipitates were resuspended in 6x LB and analyzed byWestern blotting.

IVK assays. The in vitro kinase (IVK) assays were performed with eitherimmunocomplexes from transfected cells or bacterially expressedrecombinant GST fusion proteins. For IVK assays from immunocomplexes,cell lysates were prepared in lysis buffer C (50 mM HEPES [pH7.4], 75 mM NaCl, 1 mM EDTA, 1% NP-40, PIC, and PI). Cell lysateswere incubated overnight at 4°C with anti-HA bound to proteinG-Sepharose beads. Immunocomplexes were washed twice with bufferC with 0.1% NP-40 and twice with buffer C without NP-40. Anti-HAimmunocomplexes were split into two aliquots, one for Westernblotting and the other for IVK assay. Immunocomplexes were washedtwice with kinase buffer (50 mM HEPES [pH 7.4], 0.5 mM dithiothreitol,5 mM MgCl₂, 5 mM MnCl₂) and incubated for 20 min at 30°Cin 30 µl of kinase buffer with a final concentration of25 µM ATP and [ ${gamma}$ -³²P]ATP (1 x 10^–2 µCi/pmol).For GST fusion proteins, 0.5 to 1 µg of eluted GST-Spry2or its derivatives was incubated for 20 min at 30°C in 40µl of kinase buffer with 50 µM ATP, [ ${gamma}$ -³²P]ATP (1x 10^–3 µCi/pmol), and 50 ng of purified GST-DYRK1A.Reactions were stopped by adding 6x LB, and samples were resolvedby SDS-PAGE and then stained with Coomassie blue. ³²P incorporationwas detected by autoradiography of dried gels.

Luciferase reporter assay. FGF-induced Erk-dependent transcriptional activation was measuredby an Elk-1 reporter assay. HEK-293 cells were cotransfectedwith pSG.Gal-Elk1, pE1bGal-Luc, and the Renilla expression vectorpRL-Null, together with effector plasmids. After 24 h, cellswere serum starved (Dulbecco's modified Eagle's medium supplementedwith 0.5% FCS) for 20 h, and then FGF2 (Peprotech) was addedfor 6 h. Each transfection point was done in triplicate. Luciferaseand Renilla activities were determined in cell lysates by thedual-luciferase reporter assay system (Promega) and analyzedfor bioluminescence with an AutoLumat LB 953 luminometer (BertholdTechnologies).

Statistical analysis. Significant differences between experiments were determinedby the two-tailed Student t test. Data are presented as themeans ± standard errors of the means (SEM) of independentexperiments.

RESULTS

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RESULTS

DYRK1A interacts with Spry2. To identify partners involved in the possible effects of DYRK1Aon signaling pathways, we performed a yeast two-hybrid screenof a human fetal brain cDNA library, using full-length DYRK1Aas bait. One of the putative DYRK1A-interacting proteins identifiedwas Spry2, a well-known antagonist of RTK signaling (37). Toverify the interaction between DYRK1A and Spry2 in vivo, mammaliancells were transfected with plasmids encoding full-length HA-taggedDYRK1A and Flag-tagged Spry2. Cell extracts were subjected toimmunoprecipitation with anti-HA or anti-Flag antibodies, andthe presence of the proteins in the immunoprecipitates was analyzedby Western blotting. Flag-Spry2 was coimmunoprecipitated withHA-DYRK1A in anti-HA immunocomplexes but was not precipitatedby anti-HA when expressed alone, thus indicating specific interactionbetween the two proteins (Fig. 1A). The complementary resultwas obtained when immunocomplexes were prepared with anti-Flag(Fig. 1B). These results thus demonstrate that DYRK1A and Spry2can interact in vivo in mammalian cells.

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FIG. 1. DYRK1A interacts with the Spry family of RTK modulators. (A and B) HEK-293T cells were transfected with expression plasmids encoding HA-DYRK1A and Flag-Spry2, as indicated. At 48 h posttransfection, cell lysates were subjected to immunoprecipitation with anti-HA (A) or anti-Flag (B) antibodies. The presence of the large immunoglobulin (IgG_H) is shown. (C) Schematic representation of the human Spry family members used in panels D and E. SRD, serine-rich domain. (D) Soluble extracts from cells expressing HA-DYRK1A and one of the four members of the mammalian Spry family, as indicated, were immunoprecipitated with anti-Flag, and DYRK1A and Spry proteins were detected in the complexes by Western blotting. (E) Cells were transfected as indicated with expression plasmids encoding HA-DYRK1A; Myc-Spry2; and either Flag-Spry2 (S2), Flag-Spry3 (S3), or Flag-Spry4 (S4), and cell extracts were subjected to immunoprecipitation with anti-Flag. (F) Cell lysates expressing HA-DYRK1A, Flag-Spry2, and Myc-Spry2 were subjected to immunoprecipitation with anti-Flag (IP1). The immunocomplexes were eluted from the beads with excess Flag peptide (peptide elution) and reimmunoprecipitated with anti-HA antibody (IP2). In all panels (A to F), the presence of the different proteins in both the immunoprecipitates (IP) and the total cell lysates was analyzed by Western blotting with the indicated antibodies. The positions of marker proteins (in kDa) are indicated in this and the other figures.

DYRK1A interacts with all mammalian Spry family members. The human Spry family is composed of four members, Spry1, Spry2,Spry3, and Spry4, which have a high amino acid identity in theC-terminal region, in particular within the so-called cysteine-richdomain (CRD) (38) (Fig. 1C). We wondered therefore whether DYRK1Amight interact with other Spry proteins. Coimmunoprecipitationassays confirmed that DYRK1A is able to interact with all knownmammalian Spry proteins (Spry1 to Spry4) (Fig. 1D), indicatingthat DYRK1A is a common partner of the mammalian Spry family.

Spry proteins have been reported to form active homo- and heterodimers(22, 40). We therefore tested whether the interaction of DYRK1Awith Spry family members affected their ability to form heterodimerswith Spry2. In coimmunoprecipitation experiments, the presenceof DYRK1A did not produce significant differences in the bindingof Spry2 to other Spry family members (Fig. 1E). Thus, DYRK1Abinding does not appear to interfere with heterodimer formation,and moreover, these results suggest that DYRK1A might interactwith Spry dimers. To check this possibility, we performed adouble round of immunoprecipitations, combining different epitope-taggedproteins and different antibodies, to isolate the putative trimericcomplexes. First, we used anti-Flag to immunoprecipitate proteinsbound to Flag-Spry2; both Myc-Spry2 and HA-DYRK1A were detectedin the immunocomplexes (Fig. 1F, IP1 panel). The precipitatedproteins were then eluted with an excess of competitor Flagpeptide (Fig. 1F, peptide elution panel) and subsequently immunoprecipitatedwith anti-HA antibody; these immunocomplexes contained bothFlag-Spry2 and Myc-Spry2 (Fig. 1F, IP2 panel). These resultsprovide evidence of DYRK1A binding to at least the homodimerSpry2.

The interaction of DYRK1A with Spry2 is not affected by the activation status of the FGF pathway. The interaction of some molecular partners with Spry2 or otherSpry family members is modulated by RTK activation. This isthe case with c-Cbl, which seems to require RTK-dependent phosphorylationof Spry2 at Tyr55 for binding (16, 21, 42). Conversely, otherpartners, such as Raf kinases, can interact directly with Spryproteins, independently of the activation state of RTK-dependentpathways (43, 47). In most cases, this differential behaviorhas been analyzed in the context of FGF signaling. Furthermore,we have recently demonstrated that the phosphorylation statusof a regulatory serine residue in DYRK1A, involved in 14-3-3binding, is modulated in vivo by stimulation with FGF but notEGF (1). Therefore, to investigate whether the DYRK1A/Spry2interaction is modulated by RTK activation, we carried out coimmunoprecipitationexperiments in cells overexpressing FGFR. This approach ensuresconstitutive and chronic receptor activation independently ofligand binding and mimics the continuous activation that occursduring some developmental processes and pathogenic situations.Thus, a clear increase in the levels of phospho-Erk was detectedin cells overexpressing FGFR1, and this activation was inhibitedby the expression of wild-type (wt) Flag-Spry2 but not by thedominant-negative Flag-Spry2-Y55A (Fig. 2, anti-Pi-Erk). Thephosphorylation status on Tyr residues of Spry2-wt and Spry2-Y55Awas checked by immunoblotting with anti-phospho-Tyr antibody,and the results confirmed that the major phospho-Tyr residuein Spry2 was Tyr55 (Fig. 2, anti-PY20).

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FIG. 2. DYRK1A interaction with Spry2 is not affected by FGF pathway activation. HEK-293 cells were transfected with expression plasmids encoding FGFR1 and either the wt or the Y55A mutant version of Flag-Spry2, as indicated. At 48 h posttransfection, soluble cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibody. Both the cell lysates and the immunoprecipitates (IP) were analyzed by Western blotting with the indicated antibodies (PY20, anti-phospo-Tyr antibody; Pi-Erk, anti-phospho-Erk1/2 Thr202/Tyr204).

Overexpression of FGFR1 did not alter the detectable levelsof endogenous DYRK1A or other Spry2 partners such as c-Cbl andB-Raf (Fig. 2, lysates). Interaction of c-Cbl with Spry2-wtwas detected only in FGFR1-overexpressing cells, and no interactionwas detected with Spry2-Y55A (Fig. 2, anti-Cbl in IP). In contrast,interaction of Spry2 with B-Raf was detected with both Spry2-wtand the mutant Spry2-Y55A and was independent of the activationstatus of the pathway (Fig. 2, anti-B-Raf in IP). Similarlyto B-Raf, endogenous DYRK1A protein was present in Spry2 immunocomplexesin all situations analyzed (Fig. 2, anti-DYRK1A in IP), thusindicating that DYRK1A interacts with Spry2 independently ofphosphorylation on Tyr55 and that the activation status of theFGF pathway very likely does not affect the interaction.

DYRK1A is able to interact with Spry2 through their CRD. To define the region in Spry2 responsible for interaction withDYRK1A, we performed a yeast two-hybrid screen with DYRK1A asbait and several Spry2 deletion mutants as prey. This screenidentified a region in Spry2 spanning amino acids 164 to 255as necessary for the interaction with DYRK1A (see Fig. S1A inthe supplemental material). These results were further testedby in vitro pull-down assays (see Fig. S1B in the supplementalmaterial). To confirm the interaction data, we generated a deletionmutant of Flag-Spry2 lacking amino acids 164 to 255 (Flag-Spry2 ${Delta}$ 164-255;scheme in Fig. 3B) and assayed its ability to bind DYRK1A invivo. Compared with that of full-length Spry2, interaction ofthis mutant with DYRK1A was greatly compromised (Fig. 3A). Quantificationof several experiments yielded a reduction in binding of about70% (Fig. 3C). The deleted region overlaps with the CRD in Spry2,thus indicating that the integrity of the CRD in Spry2 is necessaryfor interaction with DYRK1A.

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FIG. 3. DYRK1A interacts with the CRD of Spry2. (A) HEK-293T cells were transfected with plasmids encoding HA-DYRK1A and either the wt or the ${Delta}$ 164-255 deletion mutant version of Flag-Spry2, as indicated. At 48 h posttransfection, cell extracts were prepared and immunoprecipitated with anti-Flag. Lysates and immunocomplexes (IP) were analyzed by Western blotting with the antibodies indicated. (B) Schematic representation of the Spry2 proteins used in panel A. SRD, serine-rich domain. (C) The histogram presents the proportion of DYRK1A protein bound to Spry2 (DYRK1A in immunocomplexes/DYRK1A in lysates) as a percentage of the value obtained with wt Spry2, which was set at 100%. Data are means ± SEM of three independent experiments (**, P $≤$ 0.01).

The polyhistidine segment in DYRK1A is necessary and sufficient for interaction with Spry2. To identify the DYRK1A sequence responsible for binding to theSpry2 CRD, a yeast two-hybrid screen was performed with full-lengthand deletion mutant versions of DYRK1A as baits and Spry2 asprey (see Fig. S2A in the supplemental material). The screenidentified a region in DYRK1A spanning amino acids 600 to 616as being necessary for the interaction with Spry2. These resultswere confirmed by coimmunoprecipitation assays on cell extractsexpressing different DYRK1A carboxy-terminal deletion mutants(see Fig. S2B in the supplemental material). The identifiedsequence corresponds closely to the distinctive polyhistidinesegment in DYRK1A (590-HHHHGNSSHHHHHHHHHHHHH-611). Furthermore,a DYRK1A mutant in which only the polyhistidine stretch wasdeleted (DYRK1A ${Delta}$ His) was unable to interact with Spry2 (Fig.4B). Interestingly, an inactivating mutation in the DYRK1A kinasedomain did not affect the interaction (Fig. 4B; see also Fig.S2 in the supplemental material). Thus, the interaction of DYRK1Awith Spry2 requires the polyhistidine sequence but is independentof DYRK1A kinase activity.

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FIG. 4. The polyhistidine domain in DYRK1A is necessary and sufficient for interaction with Spry2. (A) Schematic representation of the DYRK1A proteins used in panel B. NLS, nuclear localization signal; KINASE, kinase catalytic domain; PEST, PEST domain; His, histidine repeat; S/T, serine/threonine-rich domain. (B) wt, kinase-dead (K-R), or histidine segment deletion ( ${Delta}$ His) versions of DYRK1A were overexpressed with Flag-Spry2 in HEK-293T cells, and the presence of the different proteins was analyzed in anti-Flag immunoprecipitates. (C) HEK-293T cells were transfected with expression plasmids encoding Flag-Spry2 and either GFP or a fusion protein of GFP and the histidine domain of DYRK1A (GFP-Hrep), and cell lysates were immunoprecipitated with anti-Flag antibody. The positions of the GFPs are shown. (D) Cells were transfected with pHA-DYRK1A and pFlag-Spry2, as indicated. Soluble cell extracts were subjected to immunoprecipitation with anti-Flag antibody either under standard conditions (–) or in the presence of 5 mM each of EDTA and EGTA (EDTA/EGTA), with or without 20 mM ZnCl₂ (EDTA/EGTA + ZnCl₂). In panels B to D, both lysates and the immunoprecipitates (IP) were analyzed by Western blotting with the indicated antibodies.

To test whether the polyhistidine segment is sufficient forDYRK1A binding to Spry2, we fused it to a heterologous protein(GFP) and assayed it for interaction. Coimmunoprecipitationassays confirmed that the fusion protein (GFP-Hrep), but notGFP alone, interacted with Spry2 (Fig. 4C), indicating thatthe histidine segment itself forms the recognition motif forSpry2 binding. To further support this conclusion, we were ableto show that interaction between DYRK1A and Spry2 is competedin a dosage-dependent manner by coexpressed GFP-Hrep fusionprotein (see Fig. S2C in the supplemental material).

Histidine and cysteine residues commonly mediate the formationof structured protein domains around a coordinating metal, asoccurs in zinc finger domains. It has been proposed that metalcoordination can also mediate interaction between separate proteins,through the formation of an intermolecular zinc finger-likestructure (24). We postulated that the interaction of DYRK1Aand Spry2 might involve the coordination of metal ions by theirrespective histidine and cysteine residues. To test this hypothesis,we performed coimmunoprecipitation assays with standard cellextracts or extracts supplemented with cation chelators (EDTA/EGTA)with or without an excess of Zn ions. As Fig. 4D shows, chelationof divalent cations severely impaired the interaction of DYRK1Aand Spry2; in addition, the presence of an excess of zinc notonly overcame the effect of the chelators but enhanced the interactionbetween DYRK1A and Spry2. This result is consistent with thehypothesis that the DYRK1A polyhistidine segment and the Spry2CRD interact by forming an intermolecular zinc finger-like structurecoordinated by metal ions.

DYRK1A and Spry2 interact in the brain and codistribute in nerve endings. Both DYRK1A and Spry2 mRNA transcripts are highly expressedin mouse brain in comparison with other organs or tissues (35,46). To examine the physiological relevance of the DYRK1A-Spry2interaction, we performed coimmunoprecipitation assays on mousebrain lysates. Spry2 was consistently detected in immunoprecipitateswith a specific antibody against DYRK1A (Fig. 5A), supportingthe idea of physiological interaction between Spry2 and DYRK1A.

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FIG. 5. DYRK1A and Spry2 are expressed in neurons and colocalized in the dendritic processes of cortical neurons. (A) Mouse brain extracts were subjected to immunoprecipitation with nonspecific rabbit IgGs (IgG) or with a specific rabbit anti-DYRK1A antibody. Both the mouse brain extract (input) and the immunoprecipitates (IP) were analyzed by Western blotting with the antibodies indicated. A cross-reacting band in the IP lanes is indicated with an asterisk. (B) Serial sections of adult mouse brain were processed for immunohistochemistry and immunofluorescence with antibodies against Spry2 (A, B, E, and F) and DYRK1A (C, D, G, and H). Coexpression of DYRK1A and Spry2 was detected in the processed tissue by optical microscopy (A, C, E, and G) or fluorescence microscopy (DYRK1A, green [D and H]; Spry2, red [B and F]). Subpanels A and C show coronal sections of ventral hindbrain; expression of DYRK1A and Spry2 is evident in the olive nuclei (boxed areas), and coexpression in cells of this region is shown in the corresponding immunofluorescence images in subpanels B and D (arrows). Subpanels E and G show coronal sections of cerebellum, and the boxed areas highlight coexpression of DYRK1A and Spry2 in the deep nuclei; cellular coexpression in this region is shown in the corresponding immunofluorescence images in subpanels F and H (arrows). Bars, 25 µm. (C) Double labeling of mouse primary cortical neurons for DYRK1A and Spry2, as indicated. A merged image is also shown (bottom panel). Arrows indicate neural growth cone-like structures where DYRK1A and Spry2 colocalize. Insets show a magnification of one of these structures. Bars, 25 µm.

Interaction between components of intracellular signaling pathwaysis clearly only of biological relevance if they are naturallyexpressed in the same cells. DYRK1A protein distribution hasbeen studied in detail in the mouse adult brain (35), but theprotein distribution of Spry2 in this tissue has not been reportedpreviously. To identify sites of potential physiological DYRK1A/Spry2interaction, we analyzed Spry2 expression by immunohistochemistryin brain structures where DYRK1A is known to be expressed. Asummary of the results is presented in Fig. S3 in the supplementalmaterial. In general, Spry2 immunostaining was detected throughoutthe brain, weakly in the neuropile and more strongly in cellsomas and proximal projections. Although a complete overlapin DYRK1A and Spry2 expression patterns was not apparent, themain structures showing strong DYRK1A staining were also expressingSpry2. Figure 5B shows two illustrative examples. In hindbrain,Spry2 protein was detected in cells of the olive nuclei, whereDYRK1A is also present (Fig. 5B, subpanels A and C). Coexpressionwas also seen in the deep nuclei of the cerebellum (Fig. 5B,subpanels E and G). A more detailed analysis by double immunofluorescencerevealed that the two proteins are coexpressed in the majorityof cells in these two structures (Fig. 5B, subpanels B, D, F,and H), confirming that DYRK1A and Spry2 are present in thesame cells in specific structures of the adult brain.

To gain further details about the subcellular localization ofboth proteins in the central nervous system, we performed doublecolocalization studies in dissociated cortical neuron cultures.At 10 days in vitro, these cells are already mature, althoughnumerous growth cone-like structures are still present (Fig.5C). Using specific antibodies, we detected the presence ofDYRK1A and Spry2 in the soma of cortical neurons in culture,not only in the cytosol but also in the nucleus (Fig. 5C). DYRK1Aand Spry2 were also present across the neural processes, witha punctate staining that appeared enriched in the growth cone-likestructures (Fig. 5C). At higher magnification, the overlay ofred and green fluorescence revealed strong colocalization inthe growth cones (Fig. 5C, lower panel). These results pointto a high likelihood for a functional interaction of DYRK1Aand Spry2 in neurons.

To get a better insight about DYRK1A and Spry2 codistributionin neural compartments, we subcellularly fractionated braintissue by using two different approaches (Fig. 6A). In the first,subcellular compartments were separated according to their isopycnicequilibrium point on a discontinuous sucrose gradient; in thesecond, they were separated by mass and solubility. The followingendogenous proteins were used as compartment markers to monitorthe reliability of the procedures: lamin B1 for nuclei, calretininfor the soluble cytosolic fraction, synaptophysin for the vesiclefraction, and SNAP-25 for the plasma membrane fraction. Thestarting material for both procedures was a nucleus-free lysate(S1 fraction) obtained from detergent-free mouse brain homogenates(Fig. 6A). As shown in Fig. 6B, DYRK1A and Spry2 were both presentin this postnuclear fraction. When the S1 fraction was fractionatedby discontinuous sucrose gradient centrifugation (Fig. 6C),DYRK1A was separated into two populations, one in the solubleprotein fractions (fractions 3 to 11) and the other in high-densityfractions (fractions 27 to 33). Spry2 also separated into twoprotein pools, one in the light vesicle fractions (fractions19 to 25) and the other codistributing with DYRK1A in the high-densityfractions (fractions 29 to 33). Notably, other proteins involvedin the FGF-MAPK signaling pathway, such as FGFR1, the adaptorprotein GRB2, and the MAPK Erk2 were also detected in thesefractions (Fig. 6C). In the second fractionation procedure,the postnuclear S1 fraction was separated by differential centrifugationinto an S2 soluble/microsomal fraction and a P2 crude synaptosomalfraction, which were further fractionated (scheme in Fig. 6A).DYRK1A was detected in the soluble cytosolic fraction (S3),and both DYRK1A and Spry2 were detected in the microsomal (P3)fraction and the crude synaptosomal fraction (P2) (Fig. 6D).In addition, both proteins were enriched in the fraction correspondingto synaptosomal plasmatic membranes (LP1) but were barely detectedin the synaptic vesicle fraction (LP2) (Fig. 6D).

Together, these results indicate that DYRK1A and Spry2 interactin brain and show that this interaction can take place closeto membranes in nerve endings, corresponding to the active centerswhere cells initiate signaling cascades in response to growthfactors.

DYRK1A is a kinase of Spry2. When analyzing the results of DYRK1A and Spry2 coexpressionexperiments, we observed changes in the electrophoretic mobilitypattern of Spry2, as shown for instance in Fig. 1B and D. Thismobility shift did not occur when Spry2 was coexpressed withthe kinase-dead or DYRK1A ${Delta}$ His DYRK1A mutants (Fig. 4B). The low-mobilitybands are sensitive to alkaline phosphatase treatment (see Fig.S4A in the supplemental material), indicating that they arethe result of phosphorylation events. All these data stronglysuggest that DYRK1A increases the phosphorylation state of Spry2.

To verify the ability of DYRK1A to directly phosphorylate Spry2,we carried out IVK assays with bacterially expressed GST-DYRK1Aas the source of enzyme and GST-Spry2 as substrate. Phosphatelabel incorporation was detected both in the DYRK1A fusion proteinas a result of autophosphorylation and in different Spry2 fusionproteins (Fig. 7A; see also Fig. S4B in the supplemental material).These experiments confirmed that DYRK1A can directly phosphorylateSpry2 and showed that the putative phosphorylated residues arelocated within the 164-residue N-terminal portion. We couldnot find any consensus DYRK1A phosphorylation motifs (RxxS/TP[23]) within this region. Nevertheless, the Spry2 amino-terminalsequence does contain a TP pair and several serine/threonineresidues preceded by arginine at position –3 (see Fig.S4C in the supplemental material). We generated GST-Spry2 mutantsin which some of these residues were individually replaced byalanine (T75A, T122A, and T126A). Whereas phosphate label incorporationin vitro was unaffected by the T122A and T126A mutations, phosphorylationof the Spry2-T75A mutant was reduced compared with that of Spry2-wt(Fig. 7B). This phosphorylatable site is conserved among allvertebrate Spry2 proteins identified to date (Fig. 7C).

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FIG. 7. DYRK1A phosphorylates Spry2 at threonine residue 75. (A) IVK assay using purified bacterially expressed GST-DYRK1A as the enzyme source and GST-Spry2-(1-164) as substrate. Unfused GST was used as a control. The samples were resolved by SDS-PAGE, and the gel was stained with Coomassie blue and analyzed by autoradiography. The positions of the phosphorylated species are indicated. Note that the high-mobility bands that are present in all lanes correspond to phosphorylation of C-terminally truncated forms of GST-DYRK1A. (B) Equal amounts of wt, T75A, T122A, and T126A versions of bacterially expressed and purified GST-Spry2-(1-164) were used in IVK assays with purified GST-DYRK1A. The samples were analyzed as for panel A. (C) The amino acid sequences of Spry2 from human (NP_005833), mouse (NP_036027), rat (NP_001012046), dog (XP_542623), chicken (NP_990131), and frog (NP_001006932) are aligned in the conserved region surrounding T75 (marked with an asterisk). Numbers indicate first and last amino acids listed. Shading indicates residues conserved in all sequences listed. The DYRK1A phosphorylation consensus sequence is also included. (D) Cells were transfected with expression plasmids encoding HA-DYRK1A wt or the kinase-dead mutant HA-DYRK1A-K179R (K-R) and wt or T75A versions of Flag-Spry2. Cell extracts were subjected to immunoprecipitation with anti-HA antibody, followed by IVK assay. Samples were resolved by SDS-PAGE and analyzed by autoradiography and by Western blotting with the indicated antibodies.

The ability of DYRK1A to phosphorylate Spry2 was also checkedin IVK assays with DYRK1A-specific immunocomplexes purifiedfrom transfected cells expressing DYRK1A and Spry2. In theseexperiments, phosphate was incorporated only into Spry2 coimmunoprecipitatedwith wt DYRK1A but not with the kinase-dead mutant (Fig. 7D,IVK). Likewise, Spry2-T75A showed a markedly reduced incorporationof radioactive phosphate in kinase assays from DYRK1A immunocomplexes(Fig. 7D, IVK), which cannot be due to differences in Spry2interaction with DYRK1A (Fig. 7D, IP anti-HA). We noticed thatthe effect of mutating Spry2 T75 in the IVK assays was smallerwhen using bacterially purified proteins (Fig. 7B) than whenusing immunocomplexes from mammalian cells (Fig. 7D). This wasdue to the existence of Spry2 residues that are phosphorylatedin the first type of assay but not in the second (data not shown).The different behavior could be explained by differences inthe specificity of the DYRK1A proteins expressed in each systemor by a restriction toward some phosphorylatable residues whenthe kinase and the substrate form a complex in the coimmunoprecipitationassay. We also observed that mutation T75A in Spry2 did notcompletely abrogate phosphate incorporation in the IVK assays(Fig. 7D), suggesting that other DYRK1A phosphorylated sitesmay exist. Altogether, these results indicate, therefore, thatSpry2 is phosphorylated directly by DYRK1A on at least Thr75.

Spry2 activity is modulated by DYRK1A. To study the functional role of this novel Spry2 phosphorylationsite, we analyzed the ability of Spry2-T75A to inhibit FGF-MAPKsignaling. No differences in the expression levels of Spry2-wtand Spry2-T75A were detected, either when expressed alone orin the presence of FGFR (Fig. 8A, lysates). Neither we did detectany significant difference in the level of tyrosine phosphorylationbetween the two proteins when coexpressed with FGFR (Fig. 8A,anti-PY20). However, compared with Spry2-wt, the nonphosphorylatableSpry2-T75A mutant induced a significantly increased inhibitionof Erk1/2 activation in FGFR1-overexpressing cells (Fig. 8A,anti-PiErk, and quantification in Fig. 8B). These findings suggestan increased inhibitory efficiency of the mutant Spry2-T75A.

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FIG. 8. Phosphorylation of Spry2 modulates its activity as an RTK negative regulator. (A) HEK-293 cells were transfected with expression plasmids encoding FGFR1 and either the wt or the T75A mutant version of Flag-Spry2, as indicated. At 48 h posttransfection, soluble cell lysates were prepared and subjected to immunoprecipitation with anti-Flag antibody. Both the cell lysates and the immunoprecipitates (IP) were analyzed by Western blotting with the indicated antibodies (PY20, anti-phospo-Tyr antibody; PiErk, anti-phospho-Erk1/2 Thr202/Tyr204). (B) The histogram presents the relative Erk activation in the lysates (ratio of phospho-Erk to total Erk2 signals) as a proportion of the activation in cells transfected with wt Spry2 (set at 1). Data are the means ± SEM of four independent experiments (***, P $≤$ 0.001). (C) Cells were cotransfected with pE1bGal4-luc, pGal4-Elk1, and pRL-Null plasmids, together with increasing amounts of expression plasmids encoding Spry2 wt (WT) and the nonphosphorylatable mutant T75A. Cells were stimulated with FGF2 (10 ng/ml) for 6 h, and luciferase activity was measured in triplicate plates. Values were corrected for transfection efficiency as measured by Renilla luciferase activity. Data are the means ± SEM of the induction of luciferase activity above the nonstimulated level from two independent experiments. (D) Total protein extracts from cells transfected with equivalent amounts of Spry2 expression plasmids as described for panel C were analyzed by Western blotting with anti-Flag.

To confirm this higher inhibitory capacity, we analyzed theability of Spry2 to reduce the transcriptional activity of Elk-1,a nuclear target of Erk1/2, in FGF-stimulated cells. The highsensitivity of this approach allows detection of the inhibitoryactivity of overexpressed Spry2 at very low dosages. As Fig.8C shows, the inhibitory activity of the Spry2-T75A mutant wasmore pronounced than that of the wt protein at all protein dosestested. Western blotting confirmed that there were no differencesbetween the expression levels of wt and mutant Spry2 proteinsin transfected cells (Fig. 8D). These results suggest that thephosphorylation of Spry2 at Thr75 negatively modulates the abilityof Spry2 to antagonize FGF-MAPK signaling.

DISCUSSION

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DISCUSSION

This study provides the first demonstration of a functionalinteraction between DYRK1A and Spry2. We have demonstrated thatDYRK1A and Spry2 interact in mammalian cells when exogenouslyexpressed and, more importantly, in the physiological contextof the mouse brain. DYRK1A and Spry2 proteins are present inthe same cells of different brain structures and codistributein the same subcellular fractions, supporting the conclusionthat DYRK1A and Spry2 are truly physiological partners. Thisfinding has special relevance because, although the list ofinteracting proteins for DYRK1A has grown in recent years (fora recent review, see reference 13), most of these interactionshave been detected only between exogenously overexpressed proteins.

The binding of some Spry interactors with Spry2, or with otherSpry family members, is modulated by RTK activation. Our findingsindicate that the interaction of DYRK1A with Spry2 is independentof the activation status of the FGF pathway, similarly to whathas been reported for B-Raf (47). Interestingly, DYRK1A andB-Raf are both detected in Spry2 immunocomplexes, suggestingthat binary interactions, such as Spry2/DYRK1A or Spry2/B-Raf,are mutually supportive. Given that B-Raf is a shared partnerof both Spry family members (43, 47) and DYRK1A (29), it ispossible that a trimeric complex involving all three proteinsexists in cells.

Our current work identifies the interacting domains in DYRK1Aand Spry2 as the polyhistidine domain and the CRD, respectively.Both regions are not only necessary but also sufficient forbinding. We had previously shown that the DYRK1A polyhistidinesequence targets the kinase to the splicing factor compartment(2); however, a specific protein target for this activity hasnot yet been found. The present study therefore represents thefirst report showing that this amino acid sequence can act asa protein-protein interaction domain. There is growing evidencethat kinase substrate specificity is achieved by many mechanismsin addition to direct recognition by the catalytic site (48).One such mechanism involves interactions outside the catalyticdomain, known as docking interactions, which facilitate thediscrimination of bona fide substrates for the kinase. Giventhat Spry2 is a DYRK1A substrate and that Spry2 does not getphosphorylated when coexpressed with a DYRK1A protein lackingthe histidine stretch, it can be postulated that the polyhistidinesegment is a docking site for Spry2; moreover, an appealingpossibility is that the polyhistidine segment might act as adocking site for other DYRK1A substrates as well.

The CRD in Spry2 is highly conserved among all members of theSpry family. The conservation is not only restricted to itssequence but also functional, and several activities have beenmapped to it. For example, the motif is responsible for Sprydimerization (22), harbors the region required for interactionwith Raf kinases (43) and TESK (9), and is also required forplasma membrane localization (22, 33). CRD conservation wouldjustify the ability of DYRK1A to interact with all four mammalianSpry proteins and not just Spry2. This finding places DYRK1Aas a common partner of this family of signaling inhibitors,opening the possibility of intercepting RTK signaling at multiplelevels. Furthermore, and given that DYRK1A interacts with atleast Spry2 homodimers, our results suggest that the Spry CRDcan recruit different partners simultaneously.

The special amino acid features of the DYRK1A and Spry2 bindingregions led us to propose that the interaction occurs throughthe formation of an intermolecular zinc finger-like domain,in which metal ions are coordinated by histidine and cysteineresidues at the interacting interface. Apart from the well-establishedfunction of Zn²⁺ as an organizer of intramolecular protein structures,for example in zinc fingers, ring fingers, or FYVE domains,there are also reports showing that this transition metal actsas an intermolecular linker between subunits in protein complexes.This kind of interaction features in the binding of the cytoplasmictail of CD4 with the N-terminal region of Lck (25) and the formationof complexes between cyclin T1 and granulin (24). The findingthat Zn²⁺ is required for the DYRK1A and Spry2 interaction isconsistent with our hypothesis. It is thus easy to imagine howchanges in intracellular Zn²⁺ concentration could provide away of regulating the formation of DYRK1A-Spry2 complexes. Redoxmodifications of cysteines in Spry2 might also result in differentialbinding to DYRK1A.

Our present study is the first to show that Spry2 protein isexpressed in several areas of the central nervous system inthe mouse. In some of these areas, both Spry2 and DYRK1A arecoexpressed. Moreover, proposals that DYRK1A and Spry2 interactphysiologically in the brain are strengthened by the fact thatthe proteins codistribute in the same subcellular fractionsand colocalize in the growth cone-like structures of corticalneurons in culture. The biochemical fractionation studies haverevealed that both DYRK1A and Spry2 distribute in fractionsenriched in molecules clearly involved in RTK signaling suchas FGFR, the adaptor GRB2, or the MAPK Erk2. In addition, theanalysis showed that both Spry2 and DYRK1A are present in thecrude synaptosomal fraction, preferentially associated withthe synaptic plasma membranes. Whereas Spry2 has been reportedto localize to the plasma membrane through palmitoylation and/orcaveolin binding (7, 28), nothing about DYRK1A membrane associationhas been reported or inferred from its primary structure. DYRK1Adistribution in membrane fractions should depend therefore onits interaction with specific partners, one of them being Spry2.It is also worth mentioning that a small proportion of bothproteins was detected in the free vesicle-containing fraction.This may indicate their presence in transport vesicles presentin this fraction and is consistent with evidence that Spry proteinsare associated with cytosolic vesicles (21, 28, 30) involvedin endocytic events controlling receptor trafficking (30). Inthis context, DYRK1A has been shown to phosphorylate severalcomponents of the endocytic machinery, such as dynamin, synaptojanin,and amphiphysin I (39). Taken together, these findings wouldargue that DYRK1A/Spry2 interaction has the potential to contributeto the regulation of intracellular receptor trafficking. Thissuggestion is particularly interesting in the context of theregulation of intracellular signaling in neurons, in which thepropagation of the signals received at axon terminals relieson the retrograde transport of signaling molecules to cell bodies(for a recent review, see reference 27).

Although Spry2 has been extensively reported as a cytosolicprotein in cell lines (see, for instance, references 28 and30), we have noticed that, apart from Spry2 being present inthe neuritic processes, a fraction of Spry2 is detected in theneuronal cell nucleus. This has been observed by immunofluorescenceanalysis in brain sections as well as in dissociated culturesof cortical neurons. A similar staining pattern has been reportedin primary cultures of cerebellar granule neurons (18), pointingto a not-yet-known nuclear role for Spry2.

An important finding of this work is that DYRK1A is able todirectly phosphorylate Spry2. It is known that Spry2 activityis posttranslationally regulated by phosphorylation (22, 32),which modulates several aspects of its function; however, onlytwo serine/threonine protein kinases have been described tofunctionally interact with Spry2. Phosphorylation by Mnk1 onSer112 and Ser121 controls Spry2 degradation (10). In contrast,the alteration of the Spry2 phosphorylation status by TESK isdue not to its kinase activity but to a displacement of Spry2from binding to the phosphatase PP2A (9). Here, we have demonstratedthat Spry2 is a substrate of DYRK1A and identified Thr75 asone of the residues phosphorylated by this kinase. Mutationof this threonine yields a Spry2 protein with an enhanced abilityto inhibit Erk activation upon FGF signaling. Furthermore, wehave also shown that phosphorylation of Thr75 impairs Spry2inhibitory capacity without altering Tyr55 phosphorylation levels.

All together, our findings support a role for DYRK1A as a positivemodulator of the FGF-dependent signaling pathway by antagonizingthe activity of Spry2. It is worth mentioning that related phenotypeshave been described in mouse models of increased expressionof Spry2 or decreased expression of DYRK1A. Thus, in agreementwith the proposal that the processes that shape the midbrainand cerebellum require distinct levels of FGF signaling (34),expression of a Spry2 gain-of-function allele in the embryonicmidbrain results in the absence of the inferior colliculus (4).Interestingly, this phenotype is remarkably similar to thatobserved in Dyrk1A heterozygous mice that present a marked macroscopicreduction of the mesencephalic tectum (17). It is thus possiblethat the interplay between DYRK1A and the negative regulatorSpry2 helps to ensure the appropriate strength and durationof the FGF-dependent signal.

ACKNOWLEDGMENTS

We are especially grateful to M. L. Arbonés (CRG, Barcelona,Spain) for providing adult mice. We thank A. Raya and E. Ramírezfor technical assistance, E. Martí for her advice onthe immunohistochemical analysis, E. Balducci and M. J. Ballarobrefor providing mouse primary cortical cultures, and S. Bartlettfor English editorial work. We also thank G. R. Guy, E. Nishida,and P. E. Shaw for several of the plasmids used in the study.

This group is founded by the Centro de InvestigaciónBiomédica en Red de Enfermedades Raras (CIBERER), ISCIII.S.A. and A.L. were FI predoctoral fellows (DURSI, Generalitatde Catalunya), and M.A. was a fellow of the Spanish Ministryof Health (BEFI Program). This work is supported by grants fromthe Spanish Ministry of Education and Science (BFU2004-01768and BFU2007-61043/BMC) and the European Commission AnEUploidygrant.

FOOTNOTES

* Corresponding author. Mailing address: Centre de Regulació Genòmica-CRG, Dr. Aiguader, 88, 08003 Barcelona, Spain. Phone: 34 93 316 0144. Fax: 34 93 316 0088. E-mail: susana.luna{at}crg.es

Published ahead of print on 4 August 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Department of Medical Oncology, UniversityMedical Center (UMC Utrecht), Utrecht, The Netherlands.

REFERENCES

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