Oncogene (2015) 34, 474–484 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

Casein kinase 1 regulates Sprouty2 in FGF–ERK signaling DGR Yim1,2,5, S Ghosh3, GR Guy2,6 and DM Virshup1,4 Sprouty2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase signaling, and is implicated as a tumor suppressor. SPRY2 inhibits FGF–RAS–ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2–SOS (son of sevenless) complex that transduces signals from FGFR to RAS. SPRY2 binding to GRB2 is modulated by phosphorylation but the key regulatory kinase(s) are not known. Prior studies identified the frequent presence of CK1 phosphorylation motifs on SPRY2. We therefore tested if CK1 has a role in SPRY2 phosphorylation and function. Loss of CK1 binding and inhibition of CK1 activity by two structurally distinct small molecules abrogated SPRY2 inhibition of FGF–ERK signaling, leading to decreased SPRY2 interaction with GRB2. Moreover, CK1 activity and binding are necessary for SPRY2 inhibition of FGF-stimulated neurite outgrowth in PC12 cells. Consistent with its proposed role as an inhibitor of FGF signaling, we find that CSNK1E transcript abundance negatively correlates with FGF1/FGF7 message in human gastric cancer samples. Modulation of CK1 activity may be therapeutically useful in the treatment of FGF/SPRY2-related diseases. Oncogene (2015) 34, 474–484; doi:10.1038/onc.2013.564; published online 27 January 2014 Keywords: growth factor signaling; SPRY; casein kinase 1; neurite outgrowth; gastric cancer

INTRODUCTION Receptor tyrosine kinase (RTK) signaling governs key cellular functions including proliferation and differentiation, migration, survival and metabolism (reviewed in Lemmon and Schlessinger1 and Casaletto and McClatchey2). Regulatory mechanisms and feedback controls exist in the RTK pathways to ensure specificity in cell signaling and biological outcomes. The Sprouty (SPRY) family of proteins (SPRY1 through 4) are such feedback inhibitors.3–5 SPRY2, by modulating RTK pathways, controls diverse biological processes such as neurite outgrowth6,7 and tracheal branching.8–10 Conversely, aberrant control of signaling by SPRY leads to pathological conditions. SPRY2 is downregulated in breast, liver and prostate cancers11–13 and knockout of the various Spry genes causes a range of developmental disorders in mice (reviewed in Edwin et al.14 and Guy et al.15). SPRY2 inhibits the FGF–RAS–ERK signaling pathway. Fibroblast growth factor (FGF) binding to FGF receptors (FGFR) results in receptor dimerization, phosphorylation and recruitment of the adaptor protein FGFR substrate 2 (FRS2) (reviewed in Lemmon and Schlessinger1). Growth factor receptor bound protein 2 (GRB2) subsequently transduces signals from FRS2 to son of sevenless (SOS). The Src homology 2 (SH2) domain of GRB2 docks to phosphorylated tyrosines on FRS2, whereas the Src homology 3 (SH3) domain of GRB2 recognizes proline rich sequences on SOS. Binding of GRB2 to SOS activates RAS by stimulating the SOS RAS–GEF (guanine nucleotide exchange) activity. The activated RAS subsequently signals to the RAF–MEK–ERK MAPK module (reviewed in Turner and Grose16). SPRY2 is a potent feedback inhibitor of FGF–RAS–ERK signaling.3,5,8,17 Upon FGFR1 activation, a cryptic PxxPxR motif on C-terminal SPRY2 is revealed, enabling it to bind to the SH3 domain of GRB2.17 SPRY2 binding

sequesters GRB2 from SOS, thus disconnecting signal transduction upstream of RAS.17,18 SPRY2 is a phosphoprotein and its phosphorylation undergoes complex changes during FGF signaling. Although global 32 P-orthophosphate incorporation into SPRY2 did not change significantly after growth factor stimulation,4 the sites of phosphorylation as assessed by mass spectrometry analysis changed markedly during FGFR1 activation.19 A subset of serine and threonine residues are phosphorylated by an unknown kinase(s), whereas other sites are dephosphorylated as a consequence of enhanced protein phosphatase 2 A (PP2A) interaction with SPRY2.19 It therefore appears that serine/ threonine (S/T) kinases act in tandem with PP2A (or other S/T phosphatases) to unmask the PxxPxR motif, leading to SPRY2 binding to GRB2 and the subsequent inhibition of FGF–ERK signaling. Although several kinases have been identified that phosphorylate SPRY2,20–22 little is known about their effect on SPRY2 function. A number of phosphorylation sites identified by both mass spectrometry and mutational analysis19 conform to wellestablished CK1 consensus sites of the form pS/T-X-X-S/T.23 Similar phosphorylation sites have been reported in a number of physiologic CK1 substrates including SV40 large T antigen,24 PER2,25 APC26 and NFAT1.27 In SPRY2, several of the putative CK1 sites were phosphorylated in the resting, unstimulated state,19 consistent with constitutive interaction of SPRY2 with CK1. This suggested a potential role for CK1 in SPRY2 regulation. Here we report that endogenous CK1 isoforms interact with and regulate SPRY2 in a phosphorylation-dependent manner. Using CK1 inhibitors and various SPRY2 mutants, we find that CK1

1 Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore; 2Signal Transduction Laboratory, Institute for Molecular and Cellular Biology, Biopolis, Singapore; 3Center for Computational Biology, Duke-NUS Graduate Medical School, Singapore, Singapore; 4Department of Biochemistry, YYL School of Medicine, National University of Singapore, Singapore, Singapore and 5Genome Institute of Singapore, Biopolis, Singapore. Correspondence: Professor DM Virshup, Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. E-mail: [email protected] 6 GRG is now retired. His current address is 20 Denai Endau 7, George Town, Pulau Pinang, Penang, Malaysia. Received 30 September 2013; revised 14 November 2013; accepted 10 December 2013; published online 27 January 2014

Sprouty2 requires casein kinase 1 DGR Yim et al

475 RESULTS CK1 and SPRY2 interact in a phosphorylation-dependent manner The S/T rich domain of SPRY2 has several CK1 consensus phosphorylation motifs of the form pS/T-X-X-S/T (Figure 1a). As several of these sites are phosphorylated in unstimulated cells,19 we tested whether SPRY2 interacts with CK1. SPRY2 coimmunoprecipitated with both endogenous and ectopically expressed CK1e, d and a, and this interaction was increased by the co-expression of FGFR1 (Figures 1b–e). Endogenous CK1e and d interaction with SPRY2 increased 440%, whereas CK1a binding

activity is required for SPRY2 inhibition of FGF–ERK signaling. Phosphorylation of SPRY2 by CK1 enables the binding of SPRY2 to GRB2. CK1 is physiologically relevant in the RTK–MAPK pathway, as we find that CK1 activity is required for SPRY2 to inhibit FGF/ NGF-stimulated neurite outgrowth in PC12 cells. In gastric cancers with high FGF1 or FGF7, CK1e gene (CSNK1E) expression is low, consistent with its proposed role as an inhibitor of the FGF–RAS– MAPK pathway. Therapeutic CK1 inhibition may have the unintended consequence of inhibiting SPRY2 function and therefore enhancing growth factor signaling.

c-CBL STR (S/T rich) Y55 region

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Figure 1. Casein kinase 1 interacts with SPRY2. (a) Schematic representation of SPRY2 with highlighted residues in the S/T rich region. (b) Flagtagged SPRY2 was tested for interaction with endogenous CK1e. The various constructs were expressed in cells for 16–18 h. Cells were lysed 16–18 h after transfection of the indicated constructs, followed by immunoprecipitation of the indicated proteins. (c) Endogenous CK1d immunoprecipitates (IPs) were tested for Flag-tagged SPRY2. (d) Endogenous CK1a was analyzed for interaction with Flag-tagged SPRY2. (e) Flag-tagged SPRY2 was overexpressed and Flag-IPs were tested for HA-tagged CK1s. (f ) Kinase-dead D128N CK1e was analyzed for binding to SPRY2. (g) Kinase-dead K38R CK1d was tested for binding to SPRY2. (h) Flag-tagged SPRY2 was expressed in cells. Cells were then treated with DMSO or PF670, and subsequently with Calyculin A to stimulate SPRY2 phosphorylation. Endogenous CK1e was immunoprecipitated and analyzed for interaction with Flag-tagged SPRY2. HEK293 cells were used in the above experiments. * Indicates IgG heavy chain. WCL, whole cell lysate. Numbers at the left side of blot scans indicate molecular weight reference markers in kDa. & 2015 Macmillan Publishers Limited

Oncogene (2015) 474 – 484

Sprouty2 requires casein kinase 1 DGR Yim et al

476 K38A in CK1d,31 each markedly reduced the ability of CK1 to bind to SPRY2 (Figures 1f and g). Correspondingly, kinase-dead CK1 was unable to alter the electrophoretic mobility of SPRY2. Consistent with a role for phosphorylation in the CK1–SPRY2 interaction, CalA treatment for 15 min markedly increased the interaction of endogenous CK1 with SPRY2 in HEK293 cells (Figure 1h). Pretreatment with PF670 before CalA treatment prevents the increase in CK1–SPRY2 interaction (Figure 1h, compare lanes 3, 6 and 9). The specific phosphorylation events required for CK1–SPRY2 interaction have not yet been identified.

doubled with FGFR1 signaling (quantitation in Supplementary Figures 1D–F). We next tested whether CK1 phosphorylates SPRY2 in cells. It has been previously established that SPRY2 phosphorylation can be monitored in part by changes in electrophoretic mobility on SDS–PAGE.4,19 Co-expressed CK1 increased the SPRY2 slower migrating band (Figure 1e), suggesting that CK1 phosphorylates SPRY2. Endogenous CK1 also appears to phosphorylate SPRY2, as treatment of cells with the phosphatase inhibitor calyculin A (CalA) increased SPRY2 mobility shift and abundance, and these effects of CalA were delayed by pretreatment with the CK1 inhibitor PF670462 (PF670) (Figure 1h, WCL compare lanes 3 and 6). We conclude that SPRY2 binds to and is phosphorylated by endogenous CK1. As PF670 is specific for CK1e and CK1d,28,29 it appears that these are the predominant SPRY2 kinases. The CK1–SPRY2 interaction is phosphorylation-dependent. Two independent inactivating mutations of CK1, D128N in CK1e30 and 1

50 60

107 132 178

CK1 interacts with two distinct regions on SPRY2 We mapped the regions of SPRY2 that interact with CK1 using a series of SPRY2 N- and C-terminal truncation mutants (Figures 2a and b and Supplementary Figure 2). We observed that two

275 301 315

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Figure 2. Casein kinase 1e binds two distinct regions on SPRY2. (a) Schematic diagram of SPRY2 truncation mutants tested for interaction with HA-tagged CK1e. ( þ indicates constructs that bind to HA-CK1e) (b) Schematic diagram of SPRY2 truncation mutants tested for interaction with endogenous CK1e. ( þ indicates constructs that bind to endogenous CK1e) (c) A SPRY2 construct lacking the first CK1e binding site (SPRY2D211–230) transiently expressed in HEK293 cells was tested for binding to endogenous CK1e by immunoprecipitation, SDS–PAGE and immunoblotting using the indicated antibodies. (d) SPRY2 constructs lacking the second CK1e binding region (SPRY2D179–192), and both binding regions (SPRY2D179–192D211–230 or SPRY2DD) were generated. Endogenous CK1e immunoprecipitates were tested for Flag-tagged SPRY2 and mutant. (e) Endogenous CK1d was analyzed for interaction with Flag-tagged SPRY2 with and without FGFR1. HEK293 cells were used in the above experiments. Oncogene (2015) 474 – 484

& 2015 Macmillan Publishers Limited

Sprouty2 requires casein kinase 1 DGR Yim et al

477 domains, SPRY2 aa 211–230 and aa 179–192, each contributed to the binding of both overexpressed and endogenous CK1e. Domain 1, aa 211–230, has a larger role in basal interaction (Figures 2c and d). Deletion of both regions (SPRY2D179– 192D211–230, hereafter referred to as SPRY2DD) gave near total abrogation of binding to the kinase both with and without FGFR1 signaling (Figure 2d, lanes 5 and 10). Similar to CK1e, CK1d interaction with SPRY2DD was also decreased, especially when FGF signaling was activated (Figure 2e, compare lanes 5 and 6). Because deletion of mutants may affect overall protein folding and function, we tested SPRY2DD activity in CK1-unrelated assays (Figure 3). Both SPRY2 and SPRY2DD translocated to membranes after bFGF stimulation (Figure 3a) and both bound to endogenous c-CBL (Figure 3b), suggesting that the SPRY2DD mutation did not hinder global SPRY2 activity. We therefore used SPRY2DD as a loss-of-CK1-binding SPRY2 mutant for further studies. CK1 is required for SPRY2 inhibition of FGF–ERK signaling SPRY2 is an antagonist of FGF-stimulated ERK activation. We investigated whether CK1 regulates this function of SPRY2. In control experiments, phosphorylated ERK peaked around 7 min

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following FGF stimulation of HEK293 cells, and as expected, the peak signal is decreased in SPRY2-transfected cells (Figure 4a). To test whether CK1 kinase activity regulates SPRY2 inhibition of FGF–ERK signal, we inhibited CK1 activity by pretreating cells with PF670 for 1 h before FGF stimulation. CK1 inhibition renders SPRY2 ineffective in inhibiting the FGF–ERK signal from 5 to 10 min post stimulation (Figures 4b and c). To confirm that this effect is due to the inhibition of CK1 rather than an off-target effect of PF670, we tested a structurally unrelated CK1 inhibitor, D4476.32 D4476 treatment similarly reverses the inhibitory effect of SPRY2 on bFGF-induced ERK activation (Figure 4d). Therefore, CK1 kinase activity is necessary for the inhibitory capacity of SPRY2 in FGF– ERK signaling. To test whether CK1 interaction with SPRY2 is important for SPRY2 function, we compared the inhibitor effect of wild-type versus SPRY2DD on ERK activation. SPRY2DD, although expressed at higher levels than wild-type SPRY2, was unable to inhibit FGF–ERK signal (Figure 4e). CK1 inhibition by PF670 in the absence of transfected SPRY2 also gave increased FGF–ERK signal (Figure 4b, compare lanes 1 and 3). Taken together, the data indicate that CK1 is required for the inhibitory action of SPRY2 on FGF signaling to ERK.

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Figure 3. SPRY2 loss-of-CK1-binding mutant retains certain properties of wild-type SPRY2. (a) Flag-tagged SPRY2DD was tested for membrane translocation in COS-1 cells, stimulated by bFGF or NGF. (b) The interaction of endogenous c-CBL and ectopically expressed Flag-tagged wildtype and mutant SPRY2, as indicated, were analyzed by immunoprecipitation and immunoblotting. FGFR1 and the CK1 inhibitor PF670 was added where indicated. Disheveled 2 (DVL2) band shift was used as a readout for PF670 activity.29 (c) Flag-tagged SPRY2DD IPs were tested for interaction with HA-tagged DYRK1A and Myc-tagged TESK1. HEK293 cells were used in (b) and (c). (* Indicates nonspecific band). & 2015 Macmillan Publishers Limited

Oncogene (2015) 474 – 484

Sprouty2 requires casein kinase 1 DGR Yim et al

478 Flag–SPRY2 (10 ng)

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Figure 4. CK1 is required for SPRY2 inhibition of bFGF–ERK signaling in HEK293 cells. (a) Time course of ERK activation after bFGF stimulation in the absence or presence of ectopic SPRY2, analyzed by SDS–PAGE and immunoblotting. (EV, empty vector.) (b) HEK293 cells were transfected with 5 ng Flag-tagged ERK2, and 10 ng Flag-SPRY2 expression plasmids or empty vector as indicated. CK1 inhibition with 1 mM PF670 for 1 h abrogates the effect of SPRY2 in inhibiting bFGF–ERK signaling at 7 min post stimulation. Asterisks indicate T-tests for significance (*P ¼ 0.035, **P ¼ 0.01, n.s., not significant). (c) PF670 prevents SPRY2 inhibition of bFGF–ERK signaling. (d) D4476 abrogates SPRY2 inhibition of bFGF–ERK signaling at 7 min post stimulation. Asterisks indicate T-tests for significance (*P ¼ 0.032, **P ¼ 0.033). (e) Flag-tagged SPRY2DD is less effective than wild-type Flag-SPRY2 in suppression of bFGF-stimulated ERK signaling. Oncogene (2015) 474 – 484

& 2015 Macmillan Publishers Limited

Sprouty2 requires casein kinase 1 DGR Yim et al

479 CK1 regulates SPRY2 inhibition of FGF/NGF neurite outgrowth Besides inhibiting FGF signaling to ERK, SPRY2 also blocks FGFand NGF-mediated processes in neuronal cells.6,7,35 SPRY2 prevents neurite outgrowth induced by FGF and NGF (Figures 6a and c, third column from left). Therefore, we examined whether CK1 regulates SPRY2 inhibition of FGF- or NGF-induced neurite outgrowth. Pretreatment of PC12 cells with CK1 inhibitors PF670 or D4476 before FGF or NGF stimulation rescued SPRY2 inhibition of neurite outgrowth (Figures 6a and c). Binding of CK1 to SPRY2 is essential for its function, as SPRY2DD expression fails to inhibit neurite outgrowth (Figures 6a and c). Inhibition of CK1 either by blocking its kinase activity or by deleting its binding site on SPRY2 restored the mean number of FGF- or NGF-stimulated neurite processes to that observed in the absence of SPRY2 (Figures 6b and d). These cell-based results are consistent with the biochemical evidence that the inhibitory effect of SPRY2 requires CK1. In addition, it demonstrates that CK1 activation of SPRY2 function can be seen in more than one cell line. We also noted that PF670 or D4476 treatment of unstimulated PC12 cells induced neurite outgrowth in PC12 cells (Figure 6e), with an approximately sixfold increase in the mean number of neurite processes (Figure 6f). This is consistent with the published observation that CK1e knockdown promotes neurite outgrowth in unstimulated TC-32 cells.36 Taken together, we propose that CK1 inhibits growth factor-stimulated neurite outgrowth by activation of SPRY2.

CK1 is required for SPRY2–GRB2 interaction At least two necessary steps have been identified for SPRY2 to inhibit FGF–ERK signaling. First, SPRY2 must translocate to the plasma membrane upon activation of growth factor signaling.3,33 Second, SPRY2 must bind to GRB2.17,18 In FGFR1 signaling, SPRY2 competes with SOS for binding to GRB2, sequestering GRB2 from SOS, thus disrupting the signaling pathway upstream of RAS and RAF.17,18 We therefore tested the role of CK1 in each of these steps. FGF-mediated SPRY2 translocation to the membrane upon was previously reported in COS cells.18,34 We find that FGF-stimulated recruiting of SPRY2 to the plasma membrane was not blocked by the deletion of CK1 binding sites on SPRY2 (using SPRY2DD) (Figure 3a). We next investigated whether CK1 activity is required for SPRY2–GRB2 interaction in the presence of FGFR1 signaling. We examined the SPRY2–GRB2 interaction in the presence of the CK1 inhibitors PF670 and D4476. Treatment with either of these CK1 inhibitors for 2 h before cell lysis abrogated SPRY2 binding to endogenous GRB2 (Figures 5a and b). CK1 binding to SPRY2 is also necessary for SPRY2–GRB2 interaction. SPRY2DD is unable to interact with endogenous GRB2 as efficiently as wild-type SPRY2 (Figure 5c). We conclude that CK1 activity and recruitment is important for SPRY2 to interact with GRB2. In addition to binding to GRB2, SPRY2 has also been reported to interact with the E3 ubiquitin ligase c-CBL, although the physiological role(s) of the SPRY2–c-CBL interaction is unclear, as the inhibitory function of SPRY2 on FGF–ERK signaling is independent of the SPRY2–c-CBL interaction.15,17 Nonetheless, we examined whether CK1 facilitates the SPRY2–c-CBL interaction. We find that neither the inhibition of CK1 activity by PF670 nor the deletion of CK1 binding on SPRY2 (SPRY2DD) abrogates FGFR1mediated c-CBL–SPRY2 binding (Figure 3b). Although CK1 regulates SPRY2–GRB2 interaction as well as SPRY2 inhibition of FGF–ERK signaling, it does not influence the SPRY2–c-CBL interaction. Other S/T kinases have been found to interact with the C-terminus of SPRY2. DYRK1A interacts with SPRY2 aa 164– 255.22 TESK1 binds to SPRY2 C-terminus (aa 179–315).21 Both DYRK1A and TESK1 may be negative regulators of SPRY2 inhibition of FGF–ERK signaling. Therefore, by using the SPRY2DD mutant, we tested whether DYRK1A and TESK1 shared the same binding regions as CK1 on SPRY2. We observe that the deletion of CK1 binding sites (SPRY2DD) does not abrogate interaction with TESK1 (Figure 3c, lane 9). The TESK1 binding region on SPRY2 is therefore likely to be distinct from that of CK1. However, binding to DYRK1A is diminished in SPRY2DD (Figure 3c, lane 7). We speculate that CK1 and DYRK1A compete for binding to SPRY2, and the CK1/DYRK1A association with SPRY2 may vary in different cellular contexts.

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CK1–SPRY2 in gastric cancers Increased autocrine and paracrine signaling by growth factors such as FGFs contribute to oncogenic signaling (reviewed in Turner and Grose16). Amplification of FGF1 has been reported in ovarian cancers,37,38 with paracrine FGF1 facilitating angiogenesis.37 In gastric cancers, FGF7 (or keratinocyte growth factor) protein and transcript have been found upregulated in fibroblasts,39 contributing to cancer cell proliferation in a paracrine manner. FGF1 and FGF7 negatively correlate with patient survival in gastric cancer40 and FGF1 expression may cause a poor response to cisplatin in ovarian cancer.38 Given the potential roles of FGF signaling in gastric cancers, as well as the function of SPRY2 in the signaling pathway, we analyzed whether there is a correlation between SPRY2, CK1 and FGF gene expression in gastric cancers. Analysis of gene expression data from 200 primary gastric cancers (a data set previously described in41,42) revealed that SPRY2 transcript levels do not correlate with levels of FGF1, FGF7 (Figures 7a and b) or FGF4 (Supplementary Figure 4A). Instead, we find that CSNK1E expression negatively correlated with that of FGF1 and FGF7 (Figures 7a and b). This correlation appears to be

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Figure 5. CK1 activity and binding is required for SPRY2 interaction with GRB2. (a, b) Cells expressing FGFR1 and Flag-tagged SPRY2 were treated with DMSO, PF670 or D4476 as indicated. Flag-IPs were tested for endogenous GRB2. (c) Flag-tagged SPRY2DD was tested for interaction with endogenous GRB2. HEK293 cells were used in the above experiments. & 2015 Macmillan Publishers Limited

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480 15.0

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Figure 6. CK1 activity and binding is required for SPRY2 inhibition of bFGF/NGF induced neurite outgrowth in PC12 cells. (a) MetaMorph quantitation of bFGF-induced neurite outgrowth with representative images of cells. Cells were transfected with Flag-tagged wild-type or SPRY2DD expression vectors. PF670 was used at 1 mM, and D4476 was used at 10 mM in all the experiments. (b) Mean number of neurite processes in PC12 cells transfected with various SPRY2 constructs under the indicated conditions. Error bars indicate the 95% confidence intervals of the mean. (c) MetaMorph quantitation of NGF induced neurite outgrowth with representative images of cells. Cells were transfected with wild-type SPRY2 or Flag-tagged SPRY2DD. (d) Mean number of neurite processes in PC12 cells transfected with various SPRY2 constructs, subjected to the indicated conditions. Error bars indicate 95% confidence intervals. (e) MetaMorph quantitation of neurite outgrowth of PC12 cells in the presence of CK1 inhibitors PF670 and D4476. (f ) Mean number of neurite outgrowths in PC12 cells with and without CK1 inhibitors. Error bars indicate the 95% confidence intervals of the mean. Additional PC12 cell images are in Supplementary Figure 3.

specific to a subset of FGFs, as CSNK1E expression did not vary with FGF4 expression (Supplementary Figure 4A). After correcting for multiple testing, we find a significant decrease in CSNK1E expression but not in CSNK1A1 or CSNK1D expression as FGF1 and FGF7 expression increases (Figures 7a and b, and Supplementary Figures 4C and 4D). Although CK1e, d and a may regulate SPRY2 and FGF signaling in cell-based assays, CSNK1E downregulation in gastric cancers with increased FGF1 and 7 expression is unique among the CK1 family, suggesting tissue-specific roles of CK1 isoforms in FGF signaling. DISCUSSION Here we demonstrate that CK1 is a key regulator of SPRY2 function in RTK signaling. CK1 binds to SPRY2 in a phosphorylationdependent manner, and phosphorylation by CK1 makes SPRY2 a more potent inhibitor of RTK signaling. SPRY2 binding to GRB2 is regulated by CK1 activity. This interaction is biologically relevant, as CK1 inhibition counteracts SPRY2 inhibition of FGF- and NGFstimulated neurite outgrowth. The downregulation of CK1e gene expression in FGF1- and FGF7-high gastric cancers suggests this mechanism functions in cancer proliferation. These findings also Oncogene (2015) 474 – 484

suggest that pharmacologic inhibition of CK1 may increase the intensity and duration of growth factor signaling in vivo. The phospho-regulation of SPRY2 function is complex.4,19 Other SPRY2-associated S/T kinases (DYRK1A and TESK1) have been previously identified. However, their activities are reported to inactivate, rather than activate, SPRY2 function.21,22 The data suggest that SPRY2-activating sites are phosphorylated by CK1, whereas inactivating sites are phosphorylated by DYRK1A and TESK1. The phosphorylation of both sets of sites is increased by phosphatase inhibitors and by mutants of SPRY2 that cannot bind to PP2A. Here we show that decreasing S/T phosphorylation of SPRY2 by CK1 inhibition, through either PF670/D4476 or mutation of CK1 binding sites, is also sufficient to decrease SPRY2–GRB2 interactions (Figures 5a–c). CK1 activity and binding are likewise necessary for the capacity of SPRY2 to inhibit FGF–ERK signaling (Figure 4) and FGF/NGF-mediated neurite outgrowths (Figure 6). As both CK1 and PP2A activate SPRY2, we suggest that CK1 is a kinase that functions in parallel with PP2A to achieve fine balance of the posttranslational modification on activated SPRY2 (Figure 8). CK1 has been implicated in diverse cellular processes including the Wnt/b-catenin pathway, hedgehog signaling, p53 tumor & 2015 Macmillan Publishers Limited

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481

Figure 7. CSNK1E negatively correlates with oncogenic FGF transcripts in human gastric cancers. Log2 values of the indicated gene expression are plotted against FGF expression quartiles (Q1 being the lowest value and Q4 the highest). P-values of difference between quartiles are indicated. (* Indicates significance; n.s., not significant) (a) CSNK1E and SPRY2 boxplots with FGF1 quartiles. (b) CSNK1E and SPRY2 boxplots with FGF7 quartiles. Similar results were obtained with a separate CSNK1E probe (Supplementary Figure 4B).

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Figure 8. Model of SPRY2 activation in the FGF–ERK pathway. Resting state SPRY2 adopts a conformation with its PxxPxR domain inaccessible for GRB2 binding. During FGF signaling, phosphorylation changes on SPRY2 due to both S/T kinases and phosphatases, such as CK1 and PP2A, facilitate the unmasking of the GRB2 interaction motif. This leads to SPRY2 sequestration of GRB2, and inhibition of FGF signaling to ERK.

suppression and circadian rhythms (reviewed in Cheong and Virshup43). This report places CK1 in the FGF–ERK pathway. Centrosome-localized CK1d is required for Wnt3a-stimulated neurite outgrowth in Ewing sarcoma-derived cell lines36 in a & 2015 Macmillan Publishers Limited

mechanism that requires DVL phosphorylation. Conversely, they found that knockdown of CK1e induced neurite outgrowths in unstimulated cells, in agreement with our finding that the CK1 inhibitor PF670 stimulated neurite outgrowth in PC12 cells Oncogene (2015) 474 – 484

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482 (Figures 6e and f). Thus, not surprisingly, CK1 has diverse roles in cells, and these activities are determined in part by the localization and interaction partners. The implication of CK1 and PP2A in modulating FGF and NGF signaling also suggests a possible mechanism of cross-talk with other pathways. CK1e was found to be activated by Wnt44 and glutaminergic signals.45 PP2A likewise may be activated by pathways such as b2 adrenergic signaling.46 In activating CK1 or PP2A, these signaling pathways may modulate downstream growth factor signaling. Among the SPRY family of proteins, the C-terminal PxxPxR GRB2 binding motif is found solely in SPRY2. This unique characteristic of SPRY2 could explain why it inhibits FGF–ERK signaling, whereas SPRY1 and 4 have negligible effects on the pathway.17 Homologous regions of the CK1 binding regions on SPRY2, aa 179–192 and 211–230, are found in other SPRY family members (Supplementary Figure 1C, yellow and bold highlights). In addition, consensus CK1 phosphorylation sites are also found in the S/T rich domains of the other SPRY family members (Supplementary Figure 1C, green highlight). We observed that SPRY1 and 4 interact with CK1e and CK1d, and SPRY4 but not SPRY1 binds to CK1a (Supplementary Figures 1A and B). Interestingly, although CK1a binding to SPRY1 is negligible, coexpression of the kinase induces mobility shifts in SPRY1 (Supplementary Figure 1A, lane 2). CK1 expression, however, did not induce significant changes in SPRY4 electrophoretic mobility (Supplementary Figure 1B). Therefore, CK1 may regulate the functions of other SPRY family members, but through different downstream mechanisms. Given the robust biochemical phenotypes seen in CK1-regulated SPRY2 function in FGF–ERK signaling and FGF/NGF-mediated PC12 neurite outgrowth assays, modulation of SPRY2 function through CK1 may assist in treating FGF-related nonmalignant diseases. The FGF signaling cascade has an in-built negative feedback mechanism through SPRY2. Therefore, the use of FGF for therapies may be biologically useful but clinically limited. We see this in the example of TAMARIS, a phase 3 FGF gene therapy trial for critical limb ischemia.47 Although TAMARIS failed in phase 3 studies with no significant reduction in deaths or the need for limb amputations, the therapy is still promising with no known safety problems. If SPRY2 feedback contributes to the failure of this therapy, CK1 inhibition may provide a solution in part to the FGF feedback loop, and may increase the efficacy of FGF therapies. In fact, many FGFs and FGFRs have other existing or proposed clinical uses in treatment of Parkinson’s disease, wound healing, hair growth, diabetes and several types of cancers (reviewed in Beenken and Mohammadi48). CK1 inhibition may be useful for combinatorial therapies with FGFs or FGFRs. However, inhibiting CK1 in FGF/FGFR-driven cancers may have the undesired effect of increased ERK activity.

MATERIALS AND METHODS Plasmid DNA constructs The wild-type SPRY constructs, mutant SPRY2 constructs (SPRY2D50–60, SPRY2 1–191, SPRY2 192–315) and pRK5–FGFR1 have been described previously.5,19,21 The truncation mutant constructs SPRY2 1–179 and SPRY2 179–315 were previously characterized.49 4HA-CK1e in pCEP4 (V405) was described by Tsai et al.50 and Meng et al.51 The additional truncation and deletion mutant constructs for this work were generated through PCR and molecular cloning methods, using the proof-reading Pfu DNA polymerase from Promega (Madison, WI, USA) and verified by sequencing. CK1e D128N was subsequently generated by site-directed mutagenesis through PCR with Pfu DNA polymerase.

purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibody against phosphorylated ERK1/2 (9106) was purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse and rabbit anti-Flag and anti-HA (F3165, F7425, H9658 and H6908, respectively), mouse Cy3conjugated anti-b-tubulin (C4585), rabbit anti-SPRY2 N-terminal (S1444) and agarose beads conjugated with anti-Flag M2 (A2220) were purchased from Sigma-Aldrich (St Louis, MO, USA). Mouse anti-b-actin (ab3280) was from Abcam (Cambridge, UK). Mouse antibodies against pan-ERK (610124) and CK1e (610446) were purchased from BD Transduction Laboratories (San Jose, CA, USA). Mouse anti-CK1d (128A) was obtained courtesy Eli Lilly, and the UT3 rabbit antiserum was raised against C-terminus of CK1a. Dylight 680 and 800 goat anti-mouse IgG (35518 and 35521, respectively), Dylight 680 and 800 goat anti-rabbit IgG (35568 and 35571, respectively) were purchased from Thermo Scientific (Waltham, MA, USA). AlexaFluor 488 (A11001) and 594 (A11005) goat anti-mouse IgG, AlexaFluor 488 (A11008) and 594 (A11012) goat anti-rabbit IgG were purchased from Life Technologies (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (A4416 and A4914, respectively) were purchased from Sigma-Aldrich.

Cell culture, transfection and reagents All cell lines were purchased from ATCC (Manassas, VA, USA). Human embryonic kidney (HEK) 293 cells were cultured in RPMI media supplemented with 10% fetal bovine serum and 2mM L-glutamine. PC12 and COS-1 cells were cultured as described previously.6,19,49 Cell transfections were conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance to manufacturer recommended protocols. The compounds/inhibitors dimethyl sulfoxide (DMSO), Calyculin A (CalA) and D4476 were purchased from Sigma-Aldrich, and PF670462 (PF670) from Tocris Bioscience (Bristol, UK). The growth factors bFGF (F0291) and NGF (N0513) were purchased from Sigma-Aldrich. The compounds/inhibitors and growth factors were used to treat cells at various concentrations and durations as described in the text.

Immunoprecipitation and western blot analyses Immunoprecipitation and immunoblotting were conducted as described previously,5,52 but with 1 mM dithiothreitol supplemented in the lysis buffer. In addition to enhanced chemiluminescence detection, primary antibody-bound immunoblots were probed with fluorescent dyeconjugated Dylight secondary antibodies and alternatively visualized with the LI-COR Odyssey imaging system (LI-COR. Lincoln, NE, USA). Protein bands from immunoblot images (from LI-COR Odyssey) or scans (from X-ray film) are analyzed and quantitated using the Image Processing and Analysis in Java (ImageJ, Public Domain. Developer: Wayne Rasband, NIH, Bethesda, MD, USA) software. Images were first processed to reduce background signal by 50.0 pixels and black–white inverted. Bands of interest were subsequently selected and measured for mean pixel intensity/signal.

PC12 neurite outgrowth assay and immunofluorescence microscopy The PC12 neurite outgrowth assay was performed, and COS-1 and PC12 cells were fixed and stained as previously described.21 Depending on the experimental design described in the text, PC12 cells were pretreated with DMSO, PF670 (1 mM) or D4476 (10 mM) for 1 h before growth factor stimulation for 3 days. A Zeiss LSM 710 upright confocal microscope (Zeiss, Oberkochen, Germany) was used to capture images of the cells.

Quantitation of neurite outgrowth PC12 cell images obtained from confocal/fluorescence microscopy were converted to 16-bit monochromatic, gray scale, single channel images before analysis using the MetaMorph Microscopy Automation & Image Analysis software by Molecular Devices (Sunnyvale, CA, USA). Representative images were used to obtain optimized parameters for cell bodies, nuclear stain and outgrowth settings on the Neurite Outgrowth application. The images in each data set were subsequently analyzed using the optimized parameters for neurite outgrowth quantitation.

Analysis of gastric cancer microarray Antibodies Rabbit antibodies against cMyc (SC789), CK1e (SC25423), cortactin (SC11408), FGFR1 (SC121), GRB2 (SC255) and DVL2 (SC13974) were Oncogene (2015) 474 – 484

Data from an Affymetrix expression array of 200 primary gastric cancers from the Gastric Cancer Project ’08, Singapore Patient Cohort (GSE15459) are normalized for background correction and transformed into & 2015 Macmillan Publishers Limited

Sprouty2 requires casein kinase 1 DGR Yim et al

483 Log2 values, previously by the Duke-NUS Center for Computational Biology.42 The following probes were selected for statistical analysis: CSNK1E_222015_at (CK1e), CSNK1E_226858_at (CK1e), CSNK1D_208774_at (CK1d), CSNK1A1_208866_at (CK1a), FGF1_205117_at, FGF4_206783_at, FGF7_205782_at and SPRY2_204011_at. Statistical analyses were carried out with JMP Statistical Discovery Software (SAS, Cary, NC, USA). Boxplots of CSNK1E, CSNK1D, CSNK1A1 and SPRY2 values were generated against quartiles of FGF1, FGF4 or FGF7 values.

CONFLICT OF INTEREST

20 21

22

23

The authors declare no conflict of interest. 24

ACKNOWLEDGEMENTS We thank Dr Permeen Yusoff and Dr Jit Kong Cheong for advice. This research was supported by the National Medical Research Council of Singapore under its STaR Award program to DMV, the A*STAR Graduate Scholarship (to DGRY) and by the Agency for Science, Technology and Research, Singapore.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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