Casein Kinase 1 Regulates Sprouty2 in FGF&Ndash;ERK Signaling

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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 sequesters GRB2 from SOS, thus disconnecting signal transduction 17,18 Receptor tyrosine kinase (RTK) signaling governs key cellular upstream of RAS. functions including proliferation and differentiation, migration, SPRY2 is a phosphoprotein and its phosphorylation undergoes survival and metabolism (reviewed in Lemmon and Schlessinger1 complex changes during FGF signaling. Although global 32 and Casaletto and McClatchey2). Regulatory mechanisms and P-orthophosphate incorporation into SPRY2 did not change 4 feedback controls exist in the RTK pathways to ensure specificity significantly after growth factor stimulation, the sites of in cell signaling and biological outcomes. The Sprouty (SPRY) phosphorylation as assessed by mass spectrometry analysis family of proteins (SPRY1 through 4) are such feedback changed markedly during FGFR1 activation.19 A subset of serine inhibitors.3–5 SPRY2, by modulating RTK pathways, controls and threonine residues are phosphorylated by an unknown diverse biological processes such as neurite outgrowth6,7 and kinase(s), whereas other sites are dephosphorylated as a tracheal branching.8–10 Conversely, aberrant control of signaling consequence of enhanced protein phosphatase 2 A (PP2A) by SPRY leads to pathological conditions. SPRY2 is downregulated interaction with SPRY2.19 It therefore appears that serine/ in breast, liver and prostate cancers11–13 and knockout of the threonine (S/T) kinases act in tandem with PP2A (or other S/T various Spry genes causes a range of developmental disorders in phosphatases) to unmask the PxxPxR motif, leading to SPRY2 mice (reviewed in Edwin et al.14 and Guy et al.15). binding to GRB2 and the subsequent inhibition of FGF–ERK SPRY2 inhibits the FGF–RAS–ERK signaling pathway. Fibroblast signaling. growth factor (FGF) binding to FGF receptors (FGFR) results in Although several kinases have been identified that phospho- receptor dimerization, phosphorylation and recruitment of the rylate SPRY2,20–22 little is known about their effect on SPRY2 adaptor protein FGFR substrate 2 (FRS2) (reviewed in Lemmon function. A number of phosphorylation sites identified by both and Schlessinger1). Growth factor receptor bound protein 2 (GRB2) mass spectrometry and mutational analysis19 conform to well- subsequently transduces signals from FRS2 to son of sevenless established CK1 consensus sites of the form pS/T-X-X-S/T.23 (SOS). The Src homology 2 (SH2) domain of GRB2 docks to Similar phosphorylation sites have been reported in a number phosphorylated tyrosines on FRS2, whereas the Src homology 3 of physiologic CK1 substrates including SV40 large T (SH3) domain of GRB2 recognizes proline rich sequences on antigen,24 PER2,25 APC26 and NFAT1.27 In SPRY2, several of the SOS. Binding of GRB2 to SOS activates RAS by stimulating the putative CK1 sites were phosphorylated in the resting, SOS RAS–GEF (guanine nucleotide exchange) activity. The unstimulated state,19 consistent with constitutive interaction of activated RAS subsequently signals to the RAF–MEK–ERK MAPK SPRY2 with CK1. This suggested a potential role for CK1 in SPRY2 module (reviewed in Turner and Grose16). SPRY2 is a potent regulation. feedback inhibitor of FGF–RAS–ERK signaling.3,5,8,17 Upon FGFR1 Here we report that endogenous CK1 isoforms interact with and activation, a cryptic PxxPxR motif on C-terminal SPRY2 is revealed, regulate SPRY2 in a phosphorylation-dependent manner. Using enabling it to bind to the SH3 domain of GRB2.17 SPRY2 binding CK1 inhibitors and various SPRY2 mutants, we find that CK1

1Program 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] 6GRG 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 activity is required for SPRY2 inhibition of FGF–ERK signaling. RESULTS Phosphorylation of SPRY2 by CK1 enables the binding of SPRY2 to CK1 and SPRY2 interact in a phosphorylation-dependent manner GRB2. CK1 is physiologically relevant in the RTK–MAPK pathway, The S/T rich domain of SPRY2 has several CK1 consensus as we ﬁnd that CK1 activity is required for SPRY2 to inhibit FGF/ phosphorylation motifs of the form pS/T-X-X-S/T (Figure 1a). As NGF-stimulated neurite outgrowth in PC12 cells. In gastric cancers several of these sites are phosphorylated in unstimulated cells,19 with high FGF1 or FGF7, CK1e gene (CSNK1E) expression is low, we tested whether SPRY2 interacts with CK1. SPRY2 co- consistent with its proposed role as an inhibitor of the FGF–RAS– immunoprecipitated with both endogenous and ectopically MAPK pathway. Therapeutic CK1 inhibition may have the expressed CK1e, d and a, and this interaction was increased by unintended consequence of inhibiting SPRY2 function and there- the co-expression of FGFR1 (Figures 1b–e). Endogenous CK1e and fore enhancing growth factor signaling. d interaction with SPRY2 increased 440%, whereas CK1a binding

c-CBL STR (S/T rich) GRB2 SH3 Y55 region PxxPxR 1 107 134 178 275 SPRY2 50 60 CRD 301 315 (Cysteine-rich domain)

SRSISTVSSGSRSSTRTSTSSSSSE 112 115

FGFR1 + + FGFR1 + + FGFR1 + + Flag–SPRY2 + + Flag–SPRY2 + + Flag–SPRY2 + + 43 - 43 - 43 - IB: α-Flag IB: α-Flag IB: α-Flag 34 - IP: -CK1 34 - IP: -CK1 34 - IP: -CK1 IB: α-CK1ε IB: α-CK1δ IB: α-CK1α 43 - 34 - 43 - 43 - 43 - IB: α-Flag IB: α-Flag IB: α-Flag 34 - IP: -IgG 34 - * 56 - 34 - IP: -IgG IP: -IgG IB: α-CK1ε IB: α-CK1δ * 56 - IB: α-CK1α 43 - 34 - 43 - 43 - 43 - IB: α-Flag IB: α-Flag 43 - IB: α-Flag 34 - 34 - 56 - 34 - IB: α-CK1ε WCL IB: α-CK1δ IB: α-CK1α 34 - 43 - WCL 43 - WCL IB: α-FGFR1 130 - IB: α-FGFR1 130 - IB: α-FGFR1 130 - α β IB: α-β-actin IB: α-β-actin IB: - -actin 1 2 3 4 1 2 3 4 1 2 3 4

Flag–SPRY2 + + + + + Flag–SPRY2 + + + 3HA–CK1 + HA–CK1 + + 4HA–CK1 + HA–CK1 D128N + + 4HA–CK1 + 56 - IB: α-HA 4HA–CK1 (1-349) + 43 - 56 - 43 - IP: -Flag α IB: α-SPRY2 IB: -HA 43 - IP: -Flag α IB: α-Flag IB: -HA 43 -

56 - IB: α-SPRY2 43 - WCL α IB: -HA 43 - WCL 43 - IB: α-β-actin IB: α-Flag 1 2 3 4 5 6 34 - 1 2 3 4 5

Flag–SPRY2 + + + PF670, 1 hr - 1 M 10 M Myc–CK1 WT + + CalA 40 nM, (min) 0 15 30 0 15 30 0 15 30 Myc–CK1 K38A + + Flag–SPRY2 + + + + + + + + + 43 - IB: α-Myc IB: α-Flag 34 - 56 - IP: -Flag IP: -CK1 43 - IB: α-CK1ε IB: α-SPRY2 43 - 43 - IB: α-Flag IB: α-Myc 56 - 34 - WCL IB: α-CK1ε WCL 43 - IB: α-SPRY2 43 - IB: α-β-actin IB: α-β-actin 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 Figure 1. Casein kinase 1 interacts with SPRY2. (a) Schematic representation of SPRY2 with highlighted residues in the S/T rich region. (b) Flag- tagged 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 doubled with FGFR1 signaling (quantitation in Supplementary K38A in CK1d,31 each markedly reduced the ability of CK1 to bind Figures 1D–F). We next tested whether CK1 phosphorylates SPRY2 to SPRY2 (Figures 1f and g). Correspondingly, kinase-dead CK1 was in cells. It has been previously established that SPRY2 phosphor- unable to alter the electrophoretic mobility of SPRY2. Consistent ylation can be monitored in part by changes in electrophoretic with a role for phosphorylation in the CK1–SPRY2 interaction, CalA mobility on SDS–PAGE.4,19 Co-expressed CK1 increased the SPRY2 treatment for 15 min markedly increased the interaction of slower migrating band (Figure 1e), suggesting that CK1 phos- endogenous CK1 with SPRY2 in HEK293 cells (Figure 1h). phorylates SPRY2. Endogenous CK1 also appears to phosphorylate Pretreatment with PF670 before CalA treatment prevents the SPRY2, as treatment of cells with the phosphatase inhibitor increase in CK1–SPRY2 interaction (Figure 1h, compare lanes 3, calyculin A (CalA) increased SPRY2 mobility shift and abundance, 6 and 9). The specific phosphorylation events required for and these effects of CalA were delayed by pretreatment with the CK1–SPRY2 interaction have not yet been identified. 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 CK1 interacts with two distinct regions on SPRY2 appears that these are the predominant SPRY2 kinases. We mapped the regions of SPRY2 that interact with CK1 using a The CK1–SPRY2 interaction is phosphorylation-dependent. Two series of SPRY2 N- and C-terminal truncation mutants (Figures 2a independent inactivating mutations of CK1, D128N in CK1e30 and and b and Supplementary Figure 2). We observed that two

1 5060 107 132 178 275 301 315 SPRY2 1

1-179 179 1 50 60 107 132 178 275 301 315 SPRY2 2 1 1-191 191 1-200 200 1-179 179 1-210 210 + 1-191 191 1-220 220 + 1-200 200 + 1-230 230 + 1-230 230 251 251-315 231 231-315 231 231-315 201 201-315 + 221 221-315 211 211-315 + 192 192-315 + 201 201-315 + 179 179-315 + 192 192-315 + 179 179-315 +

FGFR1 FGFR1 Flag–SPRY2 + + Flag–SPRY2 + + Flag–SPRY2211-230 + + Flag–SPRY2211-230 + + 43 - Flag–SPRY2179-192 + + IB: α-Flag Flag–SPRY2* + + 34 - IP: -CK1 IB: α-CK1ε 43 - 43 - IB: α-Flag 43 - IB: α-Flag IP: -CK1 34 - IB: α-CK1ε 43 - IB: α-CK1ε 43 - 43 - WCL IB: α-Flag 170 - 34 - IB: α-FGFR1 130 - IB: α-CK1ε 43 - IB: α-β-actin WCL 170 - 1 2 3 4 5 6 IB: α-FGFR1 130 -

IB: α-β-actin 1 2 3 4 5 6 7 8 9 10

FGFR1 Flag–SPRY2 + + Flag–SPRY2 + + 43 - IB: α-SPRY2 34 - IP: -CK1 α δ IB: -CK1 34 - 43 - IB: α-SPRY2 34 -

IB: α-CK1δ 34 - WCL 130 - IB: α-FGFR1 95 -

IB: α-β-actin 1 2 3 4 5 6 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 following FGF stimulation of HEK293 cells, and as expected, the the binding of both overexpressed and endogenous CK1e. peak signal is decreased in SPRY2-transfected cells (Figure 4a). To Domain 1, aa 211–230, has a larger role in basal interaction test whether CK1 kinase activity regulates SPRY2 inhibition of (Figures 2c and d). Deletion of both regions (SPRY2D179– FGF–ERK signal, we inhibited CK1 activity by pretreating cells with 192D211–230, hereafter referred to as SPRY2DD) gave near total PF670 for 1 h before FGF stimulation. CK1 inhibition renders SPRY2 abrogation of binding to the kinase both with and without FGFR1 ineffective in inhibiting the FGF–ERK signal from 5 to 10 min post signaling (Figure 2d, lanes 5 and 10). Similar to CK1e, CK1d stimulation (Figures 4b and c). To conﬁrm that this effect is due to interaction with SPRY2DD was also decreased, especially when the inhibition of CK1 rather than an off-target effect of PF670, we FGF signaling was activated (Figure 2e, compare lanes 5 and 6). tested a structurally unrelated CK1 inhibitor, D4476.32 D4476 Because deletion of mutants may affect overall protein folding treatment similarly reverses the inhibitory effect of SPRY2 on and function, we tested SPRY2DD activity in CK1-unrelated assays bFGF-induced ERK activation (Figure 4d). Therefore, CK1 kinase (Figure 3). Both SPRY2 and SPRY2DD translocated to membranes activity is necessary for the inhibitory capacity of SPRY2 in FGF– after bFGF stimulation (Figure 3a) and both bound to endogenous ERK signaling. To test whether CK1 interaction with SPRY2 is c-CBL (Figure 3b), suggesting that the SPRY2DD mutation did not important for SPRY2 function, we compared the inhibitor effect of hinder global SPRY2 activity. We therefore used SPRY2DD as a wild-type versus SPRY2DD on ERK activation. SPRY2DD, although loss-of-CK1-binding SPRY2 mutant for further studies. expressed at higher levels than wild-type SPRY2, was unable to inhibit FGF–ERK signal (Figure 4e). CK1 inhibition by PF670 in the CK1 is required for SPRY2 inhibition of FGF–ERK signaling absence of transfected SPRY2 also gave increased FGF–ERK signal SPRY2 is an antagonist of FGF-stimulated ERK activation. We (Figure 4b, compare lanes 1 and 3). Taken together, the data investigated whether CK1 regulates this function of SPRY2. In indicate that CK1 is required for the inhibitory action of SPRY2 on control experiments, phosphorylated ERK peaked around 7 min FGF signaling to ERK.

Unstimulated bFGF NGF Cortactin Flag-SPRY2

20μm 20μm 20μm Cortactin Flag-SPRY2

20μm 20μm 20μm

FGFR1 Flag–SPRY2 + + + Flag–SPRY2 + + + + Flag–SPRY2 + + + PF670 (1 M, 1 hr) + + HA–DYRK1A + + + Flag–SPRY2 + + Myc-TESK1 + + + Flag–SPRY250-60 + + IB: α-HA 95 - 130 - 95 - IB: α-c-CBL 95 - IB: α-Myc 43 - IP: -Flag IP: -Flag 43 - α IB: -SPRY2 34 - IB: α-SPRY2 34 - 43 - * α IB: α-HA 95 - IB: -SPRY2 34 - 95 - IB: α-Myc 130 - WCL IB: α-FGFR1 43 - WCL 95 - IB: α-SPRY2 34 - IB: α-DVL2 95 - IB: α-β-actin IB: α-β-actin 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 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 wild- type 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

EV (10 ng) Flag–SPRY2 (10 ng) bFGF (5 ng/ml), time (min) - 3 5 7 10 15 - 3 5 7 10 15

IB: α-pERK 43 -

43 - IB: α-panERK WCL 43 - IB: α-SPRY2 34 -

IB: α-β-actin 1 2 3 4 5 6 7 8 9 10 11 12

** DMSO PF670 n.s. bFGF (5 ng/ml) 1.6 Flag–SPRY2 (10 ng) + + 1.4 * 1.2 IB: α-pERK 43- 1.0 α 0.8 IB: -panERK 43- 0.6 43- IB: α-SPRY2 WCL pERK:panERK 0.4 0.2 IB: α-DVL2 95- 0.0 EV SPRY2 EV SPRY2 IB: α-β-actin DMSO PF670 1 2 3 4 bFGF

Flag–SPRY2 (10 ng) DMSO PF670 (1 M, 30 min) bFGF (5 ng/ml), time (min) - 3 5 10 30 60 - 3 5 10 30 60 43 - IB: α-pERK

43 - IB: α-panERK

WCL IB: α-SPRY2 43 -

IB: α-DVL2 95 -

1 2 3 4 5 6 7 8 9 10 11 12

bFGF (5 ng/ml) + + + Flag–SPRY2 + + * ** D4476 10 M, 1 hr + 1.2

1 IB: α-pERK 43 - 0.8

IB: α-panERK 43 - 0.6

0.4

43 - WCL pERK:panERK IB: α-SPRY2 0.2 0 EV SPRY2 SPRY2 IB: α-DVL2 95 - DMSO D4476

IB: α-β-actin bFGF 1 2 3 4

Flag–SPRY2 (10 ng) Flag–SPRY2 (10 ng) bFGF (5 ng/ml), time (min) 0 3 5 7 10 15 0 3 5 7 10 15

43 - IB: α-pERK

43 - IB: α-panERK

WCL 43 - IB: α-SPRY2

IB: α-β-actin

1 2 3 4 5 6 7 8 9 10 11 12 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 is required for SPRY2–GRB2 interaction CK1 regulates SPRY2 inhibition of FGF/NGF neurite outgrowth At least two necessary steps have been identified for SPRY2 to Besides inhibiting FGF signaling to ERK, SPRY2 also blocks FGF- inhibit FGF–ERK signaling. First, SPRY2 must translocate to the and NGF-mediated processes in neuronal cells.6,7,35 SPRY2 plasma membrane upon activation of growth factor signaling.3,33 prevents neurite outgrowth induced by FGF and NGF (Figures Second, SPRY2 must bind to GRB2.17,18 In FGFR1 signaling, SPRY2 6a and c, third column from left). Therefore, we examined whether competes with SOS for binding to GRB2, sequestering GRB2 from CK1 regulates SPRY2 inhibition of FGF- or NGF-induced neurite SOS, thus disrupting the signaling pathway upstream of RAS and outgrowth. Pretreatment of PC12 cells with CK1 inhibitors PF670 RAF.17,18 We therefore tested the role of CK1 in each of these or D4476 before FGF or NGF stimulation rescued SPRY2 inhibition steps. of neurite outgrowth (Figures 6a and c). Binding of CK1 to SPRY2 is FGF-mediated SPRY2 translocation to the membrane upon was essential for its function, as SPRY2DD expression fails to inhibit previously reported in COS cells.18,34 We find that FGF-stimulated neurite outgrowth (Figures 6a and c). Inhibition of CK1 either by recruiting of SPRY2 to the plasma membrane was not blocked by blocking its kinase activity or by deleting its binding site on SPRY2 the deletion of CK1 binding sites on SPRY2 (using SPRY2DD) restored the mean number of FGF- or NGF-stimulated neurite (Figure 3a). We next investigated whether CK1 activity is required processes to that observed in the absence of SPRY2 (Figures 6b for SPRY2–GRB2 interaction in the presence of FGFR1 signaling. and d). These cell-based results are consistent with the We examined the SPRY2–GRB2 interaction in the presence of the biochemical evidence that the inhibitory effect of SPRY2 requires CK1 inhibitors PF670 and D4476. Treatment with either of these CK1. In addition, it demonstrates that CK1 activation of SPRY2 CK1 inhibitors for 2 h before cell lysis abrogated SPRY2 binding to function can be seen in more than one cell line. endogenous GRB2 (Figures 5a and b). CK1 binding to SPRY2 is also We also noted that PF670 or D4476 treatment of unstimulated necessary for SPRY2–GRB2 interaction. SPRY2DD is unable to PC12 cells induced neurite outgrowth in PC12 cells (Figure 6e), interact with endogenous GRB2 as efficiently as wild-type SPRY2 with an approximately sixfold increase in the mean number of (Figure 5c). We conclude that CK1 activity and recruitment is neurite processes (Figure 6f). This is consistent with the published important for SPRY2 to interact with GRB2. observation that CK1e knockdown promotes neurite outgrowth in In addition to binding to GRB2, SPRY2 has also been reported to unstimulated TC-32 cells.36 Taken together, we propose that CK1 interact with the E3 ubiquitin ligase c-CBL, although the inhibits growth factor-stimulated neurite outgrowth by activation physiological role(s) of the SPRY2–c-CBL interaction is unclear, as of SPRY2. 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. CK1–SPRY2 in gastric cancers We find that neither the inhibition of CK1 activity by PF670 nor the Increased autocrine and paracrine signaling by growth factors deletion of CK1 binding on SPRY2 (SPRY2DD) abrogates FGFR1- such as FGFs contribute to oncogenic signaling (reviewed in mediated c-CBL–SPRY2 binding (Figure 3b). Although CK1 Turner and Grose16). Amplification of FGF1 has been reported in regulates SPRY2–GRB2 interaction as well as SPRY2 inhibition of ovarian cancers,37,38 with paracrine FGF1 facilitating FGF–ERK signaling, it does not influence the SPRY2–c-CBL angiogenesis.37 In gastric cancers, FGF7 (or keratinocyte growth interaction. factor) protein and transcript have been found upregulated in Other S/T kinases have been found to interact with the fibroblasts,39 contributing to cancer cell proliferation in a paracrine C-terminus of SPRY2. DYRK1A interacts with SPRY2 aa 164– manner. FGF1 and FGF7 negatively correlate with patient survival 255.22 TESK1 binds to SPRY2 C-terminus (aa 179–315).21 Both in gastric cancer40 and FGF1 expression may cause a poor DYRK1A and TESK1 may be negative regulators of SPRY2 response to cisplatin in ovarian cancer.38 Given the potential roles inhibition of FGF–ERK signaling. Therefore, by using the SPRY2DD of FGF signaling in gastric cancers, as well as the function of SPRY2 mutant, we tested whether DYRK1A and TESK1 shared the same in the signaling pathway, we analyzed whether there is a binding regions as CK1 on SPRY2. We observe that the deletion of correlation between SPRY2, CK1 and FGF gene expression in CK1 binding sites (SPRY2DD) does not abrogate interaction with gastric cancers. TESK1 (Figure 3c, lane 9). The TESK1 binding region on SPRY2 is Analysis of gene expression data from 200 primary gastric therefore likely to be distinct from that of CK1. However, binding cancers (a data set previously described in41,42) revealed that to DYRK1A is diminished in SPRY2DD (Figure 3c, lane 7). We SPRY2 transcript levels do not correlate with levels of FGF1, FGF7 speculate that CK1 and DYRK1A compete for binding to SPRY2, (Figures 7a and b) or FGF4 (Supplementary Figure 4A). Instead, we and the CK1/DYRK1A association with SPRY2 may vary in different find that CSNK1E expression negatively correlated with that of cellular contexts. FGF1 and FGF7 (Figures 7a and b). This correlation appears to be

FGFR1 FGFR1 FGFR1 Flag–SPRY2 + + Flag–SPRY2 + + Flag–SPRY2 + + PF670 (1 M, 2 hrs) + D4476 (10 M, 2 hrs) + Flag–SPRY2 +

26 - α 26 - 26- IB: α-GRB2 IB: -GRB2 IB: α-GRB2 43 - IP: -Flag 43 - IP: -Flag IB: α-SPRY2 IB: α-SPRY2 43- IP: -Flag IB: α-SPRY2 43 - 34- 43 - IB: α-SPRY2 IB: α-SPRY2 WCL 43- IB: α-FGFR1120 - α IB: α-DVL2 95 - WCL IB: -SPRY2 34- IB: α-β-actin WCL IB: α-FGFR1 120 - 1 2 IB: α-FGFR1 120- 95- IB: α-β-actin α β 12 IB: - -actin 1 2 3 4 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 Oncogene (2015) 474 – 484 Sprouty2 requires casein kinase 1 DGR Yim et al 480

3 100% 2.5 12.7 12.4 Number of 90% 15.0 13.5 22.9 processes 2 22.2 4 and more 80% 14.0 16.7 1.5 18.5 3 70% 12.8 1

2 processes 18.6 60% 22.9 1 Mean number of 0.5 22.1 22.9 50% 0 0 EV EV SPRY2 SPRY2 SPRY2 SPRY2ΔΔ 84.2

% of cells 40% 28.4 73.7 22.4 DMSO DMSO DMSO PF670 D4476 - 30% 28.6 26.3 - FGF FGF FGF FGF FGF 20% 26.3 25.7 10% 15.8 15.1 1.4 0% 1.2 EV EV SPRY2 SPRY2 SPRY2 SPRY2 1 DMSO DMSO DMSO PF670 D4476 - - bFGF bFGF bFGF bFGF bFGF 0.8 0.6 1.8

processes 0.4 Tubulin 1.6 Flag-Vector Mean number of 0.2 1.4 Flag-SPRY2 0 ΔΔ 1.2 Cell count 738 367 266 236 179 210 EV EV SPRY2 SPRY2 SPRY2 1 DMSO DMSO DMSO PF670 - 0.8 100% - NGF NGF NGF NGF 4.4 6.3 4.8 Number of 0.6 10.4 processes 90% 10.3 0.4 19.8 13.5 23.7 80% 4 and more 100% 0.2 5.1 6.9 Number of Mean number of processes 3 0 70% 90% processes 33.9 2 16.0 DMSO PF670 D4476 30.4 4 and more 60% 1 80% 39.6 3 50% 0 70% 47.7 44.0 2 88.3

% of cells 40% 77.4 60% 1 30% 50% 0 48.5 50.4 20% % of cells 40% 79.8 27.2 40.6 10% 30% 35.9 0% 20% EV EV SPRY2 SPRY2 SPRY2 DMSO DMSO DMSO PF670 - 10% 10.9 12.9 - NGF NGF NGF NGF 0% DMSO PF670 D4476 Tubulin Flag-Vector Flag-SPRY2 Tubulin Cell count585 789 316 237 561 Cell count 243 256 101 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 suggest that pharmacologic inhibition of CK1 may increase the with FGF4 expression (Supplementary Figure 4A). After correcting intensity and duration of growth factor signaling in vivo. for multiple testing, we find a significant decrease in CSNK1E The phospho-regulation of SPRY2 function is complex.4,19 Other expression but not in CSNK1A1 or CSNK1D expression as FGF1 and SPRY2-associated S/T kinases (DYRK1A and TESK1) have been FGF7 expression increases (Figures 7a and b, and Supplementary previously identified. However, their activities are reported to Figures 4C and 4D). Although CK1e, d and a may regulate SPRY2 inactivate, rather than activate, SPRY2 function.21,22 The data and FGF signaling in cell-based assays, CSNK1E downregulation in suggest that SPRY2-activating sites are phosphorylated by CK1, gastric cancers with increased FGF1 and 7 expression is unique whereas inactivating sites are phosphorylated by DYRK1A and among the CK1 family, suggesting tissue-specific roles of CK1 TESK1. The phosphorylation of both sets of sites is increased by isoforms in FGF signaling. 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 DISCUSSION of CK1 binding sites, is also sufficient to decrease SPRY2–GRB2 Here we demonstrate that CK1 is a key regulator of SPRY2 function interactions (Figures 5a–c). CK1 activity and binding are likewise in RTK signaling. CK1 binds to SPRY2 in a phosphorylation- necessary for the capacity of SPRY2 to inhibit FGF–ERK signaling dependent manner, and phosphorylation by CK1 makes SPRY2 a (Figure 4) and FGF/NGF-mediated neurite outgrowths (Figure 6). more potent inhibitor of RTK signaling. SPRY2 binding to GRB2 is As both CK1 and PP2A activate SPRY2, we suggest that CK1 is a regulated by CK1 activity. This interaction is biologically relevant, kinase that functions in parallel with PP2A to achieve fine balance as CK1 inhibition counteracts SPRY2 inhibition of FGF- and NGF- of the posttranslational modification on activated SPRY2 stimulated neurite outgrowth. The downregulation of CK1e gene (Figure 8). expression in FGF1- and FGF7-high gastric cancers suggests this CK1 has been implicated in diverse cellular processes including mechanism functions in cancer proliferation. These findings also the Wnt/b-catenin pathway, hedgehog signaling, p53 tumor

Oncogene (2015) 474 – 484 & 2015 Macmillan Publishers Limited Sprouty2 requires casein kinase 1 DGR Yim et al 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).

PP2A PxxPxR motif P P P Cysteine Rich Domain

S/T rich region Resting state Sprouty2 CK1 SPRY2 50-60 region

CK1 binding site

FGFR1 activation P Resting state phosphorylation

P Activated state phosphorylation

PP2A P

Activated GRB2 Sprouty2 ERK inhibition CK1 P P P P * Not to scale 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 mechanism that requires DVL phosphorylation. Conversely, they Virshup43). This report places CK1 in the FGF–ERK pathway. found that knockdown of CK1e induced neurite outgrowths in Centrosome-localized CK1d is required for Wnt3a-stimulated unstimulated cells, in agreement with our ﬁnding that the CK1 neurite outgrowth in Ewing sarcoma-derived cell lines36 in a inhibitor PF670 stimulated neurite outgrowth in PC12 cells

& 2015 Macmillan Publishers Limited Oncogene (2015) 474 – 484 Sprouty2 requires casein kinase 1 DGR Yim et al 482 (Figures 6e and f). Thus, not surprisingly, CK1 has diverse roles in purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse cells, and these activities are determined in part by the localization antibody against phosphorylated ERK1/2 (9106) was purchased from Cell and interaction partners. Signaling Technology (Beverly, MA, USA). Mouse and rabbit anti-Flag and The implication of CK1 and PP2A in modulating FGF and NGF anti-HA (F3165, F7425, H9658 and H6908, respectively), mouse Cy3- signaling also suggests a possible mechanism of cross-talk with conjugated anti-b-tubulin (C4585), rabbit anti-SPRY2 N-terminal (S1444) 44 and agarose beads conjugated with anti-Flag M2 (A2220) were purchased other pathways. CK1e was found to be activated by Wnt and from Sigma-Aldrich (St Louis, MO, USA). Mouse anti-b-actin (ab3280) was glutaminergic signals.45 PP2A likewise may be activated by 46 from Abcam (Cambridge, UK). Mouse antibodies against pan-ERK (610124) pathways such as b2 adrenergic signaling. In activating CK1 or and CK1e (610446) were purchased from BD Transduction Laboratories PP2A, these signaling pathways may modulate downstream (San Jose, CA, USA). Mouse anti-CK1d (128A) was obtained courtesy Eli Lilly, growth factor signaling. and the UT3 rabbit antiserum was raised against C-terminus of CK1a. Among the SPRY family of proteins, the C-terminal PxxPxR GRB2 Dylight 680 and 800 goat anti-mouse IgG (35518 and 35521, respectively), binding motif is found solely in SPRY2. This unique characteristic Dylight 680 and 800 goat anti-rabbit IgG (35568 and 35571, respectively) of SPRY2 could explain why it inhibits FGF–ERK signaling, whereas were purchased from Thermo Scientific (Waltham, MA, USA). AlexaFluor SPRY1 and 4 have negligible effects on the pathway.17 488 (A11001) and 594 (A11005) goat anti-mouse IgG, AlexaFluor 488 (A11008) and 594 (A11012) goat anti-rabbit IgG were purchased from Life Homologous regions of the CK1 binding regions on SPRY2, aa Technologies (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conju- 179–192 and 211–230, are found in other SPRY family members gated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (Supplementary Figure 1C, yellow and bold highlights). In (A4416 and A4914, respectively) were purchased from Sigma-Aldrich. addition, consensus CK1 phosphorylation sites are also found in the S/T rich domains of the other SPRY family members Cell culture, transfection and reagents (Supplementary Figure 1C, green highlight). We observed that All cell lines were purchased from ATCC (Manassas, VA, USA). Human SPRY1 and 4 interact with CK1e and CK1d, and SPRY4 but not embryonic kidney (HEK) 293 cells were cultured in RPMI media a SPRY1 binds to CK1 (Supplementary Figures 1A and B). supplemented with 10% fetal bovine serum and 2mML-glutamine. PC12 Interestingly, although CK1a binding to SPRY1 is negligible, co- and COS-1 cells were cultured as described previously.6,19,49 Cell expression of the kinase induces mobility shifts in SPRY1 transfections were conducted with Lipofectamine 2000 (Invitrogen, (Supplementary Figure 1A, lane 2). CK1 expression, however, did Carlsbad, CA, USA) in accordance to manufacturer recommended not induce significant changes in SPRY4 electrophoretic mobility protocols. The compounds/inhibitors dimethyl sulfoxide (DMSO), (Supplementary Figure 1B). Therefore, CK1 may regulate the Calyculin A (CalA) and D4476 were purchased from Sigma-Aldrich, and functions of other SPRY family members, but through different PF670462 (PF670) from Tocris Bioscience (Bristol, UK). The growth factors downstream mechanisms. bFGF (F0291) and NGF (N0513) were purchased from Sigma-Aldrich. The Given the robust biochemical phenotypes seen in compounds/inhibitors and growth factors were used to treat cells at various concentrations and durations as described in the text. 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 Immunoprecipitation and western blot analyses Immunoprecipitation and immunoblotting were conducted as described nonmalignant diseases. The FGF signaling cascade has an in-built 5,52 negative feedback mechanism through SPRY2. Therefore, the use previously, but with 1 mM dithiothreitol supplemented in the lysis of FGF for therapies may be biologically useful but clinically buffer. In addition to enhanced chemiluminescence detection, primary antibody-bound immunoblots were probed with fluorescent dye- limited. We see this in the example of TAMARIS, a phase 3 FGF 47 conjugated Dylight secondary antibodies and alternatively visualized gene therapy trial for critical limb ischemia. Although TAMARIS with the LI-COR Odyssey imaging system (LI-COR. Lincoln, NE, USA). failed in phase 3 studies with no significant reduction in deaths or Protein bands from immunoblot images (from LI-COR Odyssey) or scans the need for limb amputations, the therapy is still promising with (from X-ray film) are analyzed and quantitated using the Image Processing no known safety problems. If SPRY2 feedback contributes to the and Analysis in Java (ImageJ, Public Domain. Developer: Wayne Rasband, failure of this therapy, CK1 inhibition may provide a solution in NIH, Bethesda, MD, USA) software. Images were first processed to reduce part to the FGF feedback loop, and may increase the efficacy of background signal by 50.0 pixels and black–white inverted. Bands of FGF therapies. In fact, many FGFs and FGFRs have other existing or interest were subsequently selected and measured for mean pixel proposed clinical uses in treatment of Parkinson’s disease, wound intensity/signal. healing, hair growth, diabetes and several types of cancers (reviewed in Beenken and Mohammadi48). CK1 inhibition may be PC12 neurite outgrowth assay and immunofluorescence useful for combinatorial therapies with FGFs or FGFRs. However, microscopy The PC12 neurite outgrowth assay was performed, and COS-1 and PC12 inhibiting CK1 in FGF/FGFR-driven cancers may have the 21 undesired effect of increased ERK activity. cells were fixed and stained as previously described. 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, MATERIALS AND METHODS Oberkochen, Germany) was used to capture images of the cells. Plasmid DNA constructs The wild-type SPRY constructs, mutant SPRY2 constructs (SPRY2D50–60, Quantitation of neurite outgrowth SPRY2 1–191, SPRY2 192–315) and pRK5–FGFR1 have been described 5,19,21 PC12 cell images obtained from confocal/fluorescence microscopy were previously. The truncation mutant constructs SPRY2 1–179 and SPRY2 converted to 16-bit monochromatic, gray scale, single channel images 179–315 were previously characterized.49 4HA-CK1e in pCEP4 (V405) was 50 51 before analysis using the MetaMorph Microscopy Automation & Image described by Tsai et al. and Meng et al. The additional truncation and Analysis software by Molecular Devices (Sunnyvale, CA, USA). Representa- deletion mutant constructs for this work were generated through PCR and tive images were used to obtain optimized parameters for cell bodies, molecular cloning methods, using the proof-reading Pfu DNA polymerase nuclear stain and outgrowth settings on the Neurite Outgrowth applica- from Promega (Madison, WI, USA) and verified by sequencing. CK1e D128N tion. The images in each data set were subsequently analyzed using the was subsequently generated by site-directed mutagenesis through PCR optimized parameters for neurite outgrowth quantitation. with Pfu DNA polymerase. Analysis of gastric cancer microarray Antibodies Data from an Affymetrix expression array of 200 primary gastric cancers Rabbit antibodies against cMyc (SC789), CK1e (SC25423), cortactin from the Gastric Cancer Project ’08, Singapore Patient Cohort (GSE15459) (SC11408), FGFR1 (SC121), GRB2 (SC255) and DVL2 (SC13974) were are normalized for background correction and transformed into

Oncogene (2015) 474 – 484 & 2015 Macmillan Publishers Limited Sprouty2 requires casein kinase 1 DGR Yim et al 483 Log2 values, previously by the Duke-NUS Center for Computational ERK inhibition downstream of fibroblast growth factor receptor stimulation. J Biol 42 Biology. The following probes were selected for statistical analysis: Chem 2007; 282: 9117–9126. CSNK1E_222015_at (CK1e), CSNK1E_226858_at (CK1e), CSNK1D_208774_at 20 DaSilva J, Xu L, Kim HJ, Miller WT, Bar-Sagi D. Regulation of sprouty stability by (CK1d), CSNK1A1_208866_at (CK1a), FGF1_205117_at, FGF4_206783_at, Mnk1-dependent phosphorylation. Mol Cell Biol 2006; 26: 1898–1907. FGF7_205782_at and SPRY2_204011_at. Statistical analyses were carried 21 Chandramouli S, Yu CY, Yusoff P, Lao DH, Leong HF, Mizuno K et al. Tesk1 interacts out with JMP Statistical Discovery Software (SAS, Cary, NC, USA). 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2015) 474 – 484 & 2015 Macmillan Publishers Limited