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Molecular and Cellular Biology, March 2006, p. 1898-1907, Vol. 26, No. 5
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.5.1898-1907.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Regulation of Sprouty Stability by Mnk1-Dependent Phosphorylation{ddagger}

John DaSilva,1,2,{dagger} Lizhong Xu,1,{dagger} Hong Joo Kim,1,3 W. Todd Miller,4 and Dafna Bar-Sagi1*

Department of Molecular Genetics and Microbiology,1 Graduate Program in Genetics,2 Graduate Program in Molecular and Cellular Biology,3 Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 117944

Received 8 August 2005/ Returned for modification 6 September 2005/ Accepted 14 December 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sprouty (Spry) proteins are negative feedback modulators of receptor tyrosine kinase pathways in Drosophila melanogaster and mammals. Mammalian Spry proteins have been shown to undergo tyrosine and serine phosphorylation in response to growth factor stimulation. While several studies have addressed the function of tyrosine phosphorylation of Spry, little is known about the significance of Spry serine phosphorylation. Here we identify mitogen-activated protein kinase-interacting kinase 1 (Mnk1) as the kinase that phosphorylates human Spry2 (hSpry2) on serines 112 and 121. Mutation of these serine residues to alanine or inhibition of Mnk1 activity increases the rate of ligand-induced degradation of hSpry2. Conversely, enhancement of serine phosphorylation achieved through either the inhibition of cellular phosphatases or the expression of active Mnk1 results in the stabilization of hSpry2. Previous studies have shown that growth factor stimulation induces the proteolytic degradation of hSpry2 by stimulating tyrosine phosphorylation on hSpry2, which in turn promotes c-Cbl binding and polyubiquitination. A mutant of hSpry2 that is deficient in serine phosphorylation displays enhanced tyrosine phosphorylation and c-Cbl binding, indicating that serine phosphorylation stabilizes hSpry2 by exerting an antagonistic effect on tyrosine phosphorylation. Moreover, loss of serine phosphorylation and the resulting enhanced degradation of hSpry2 impair its capacity to antagonize fibroblast growth factor-induced extracellular signal-regulated kinase activation. Our results imply that Mnk1-mediated serine phosphorylation of hSpry2 constitutes a regulatory mechanism to extend the temporal range of Spry activity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growth factor signaling mediated by receptor tyrosine kinases (RTKs) controls a wide spectrum of fundamental cellular processes, including proliferation, differentiation, and survival (42, 51). Deregulation of RTK signaling has been implicated in the establishment of developmental disorders and in the progression of many types of cancer, indicating that the intensity and duration of RTK signaling must be precisely regulated to establish an appropriate biological response. In support of this notion, it has been shown that differences in the levels of RTK signaling determine the specification of distinct cell fates during Drosophila melanogaster embryogenesis (52). In addition, the extent and kinetics of RTK-induced extracellular signal-regulated kinase (ERK) activity can dictate whether mammalian cells undergo differentiation or proliferation (33).

To achieve fine-tuned regulation of RTK signaling, cells employ multiple negative feedback mechanisms that intercept RTK pathways at different levels (11, 13). One such mechanism involves the conserved antagonist of several RTKs, Sprouty (Spry). In Drosophila, Spry has been shown to interfere with both epidermal growth factor (EGF) receptor (EGFR) and fibroblast growth factor (FGF) receptor (FGFR) signaling either at the level of the receptor itself or by preventing Ras-mediated activation of ERK (5, 17, 28, 43). Four orthologs of Drosophila Spry (dSpry) have been identified in mammals (reviewed in references 16 and 26), with Spry2 showing the highest sequence homology to the Drosophila counterpart. Similarly to dSpry, mammalian Spry2 functions as a negative feedback inhibitor of RTK signaling during organogenesis (6, 32, 35, 53, 57, 59). In cultured cells, the expression of Spry2 inhibits FGF- and vascular endothelial growth factor (VEGF)-induced proliferation, differentiation, and migration by interfering with Ras or Raf activation (15, 23, 29, 50, 65). Although the molecular basis of this interfering effect is not completely understood, several studies have suggested that it might arise as a result of the capacity of Spry to sequester positive regulators or facilitate the action of negative regulators of the RTK-Ras-ERK cascade (20, 49, 58, 65).

Ligand-mediated activation of EGFR or FGFR has been shown to induce an increase in Spry transcripts in cultured cells (23, 38, 50), and a close spatial and temporal relationship between RTK signaling and Spry gene expression has also been observed in various mammalian embryonic tissues, including lung, limb bud, brain, kidney, digestive tract, heart, and muscle (1, 8, 32, 35, 59, 66). In Drosophila, Spry expression during embryonic development coincides with known sites of RTK signaling (5, 17, 28, 43). Thus, in a manner similar to that of other feedback regulators of RTK pathways, the inhibitory loop that is set up by the expression of Spry is initiated by signal-dependent engagement of the transcriptional machinery. In addition, we and others have shown that the accumulation of Spry can also be controlled at the posttranslational level through the following sequence of events. Growth factor stimulation induces the phosphorylation of Spry2 on a conserved tyrosine residue (Tyr 55), which in turn promotes the binding of the E3 ubiquitin ligase c-Cbl to Spry2. As a consequence, human Spry2 (hSpry2) undergoes polyubiquitination and proteasomal degradation (12, 18, 47). This pathway has been postulated to serve as a mechanism to control the duration of Spry activity (26, 46).

In this study, we have found that the levels of Spry are subject to regulation by another posttranslational modification involving serine phosphorylation. We demonstrate that growth factor-induced serine phosphorylation of hSpry2 leads to the stabilization of the protein. Serine phosphorylation of hSpry2 occurs on residues 112 and 121 and is mediated by mitogen-activated protein kinase (MAPK)-interacting kinase 1 (Mnk1). A serine phosphorylation-deficient mutant of hSpry2 displays enhanced RTK-mediated tyrosine phosphorylation and an accelerated rate of proteolytic degradation. These observations indicate that the balance between serine and tyrosine phosphorylation of Spry may dictate the temporal properties of the feedback regulation of RTK signaling.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and reagents. Constructs encoding hemagglutinin (HA)-tagged wild-type and Y55F mutant hSpry2 and HA-tagged ERK2 in the pCGN vector were described previously (9, 18). Constructs of hSpry2 mutants S112A, S121A, S112A/S121A, and Y55F/S112A/S121A were generated by PCR site-directed mutagenesis. The bacterial expression constructs encoding His-tagged hSpry21-172aa, hSpry2-S112A1-172aa, hSpry2-S121A1-172aa, and hSpry2-S112A/S121A1-172aa were generated by cloning the respective hSpry2 fragments into the BamHI-EcoRI sites of the pProExHTb vector (Invitrogen). The construct encoding T7-tagged protein phosphatase 2A (PP2A)-C{alpha} was generated by cloning human PP2A-C{alpha} cDNA (kindly provided by David Brautigan, University of Virginia) into the pCGT vector. All engineered constructs were confirmed by sequencing. Expression plasmids encoding EGFR and c-Cbl were kindly provided by Joseph Schlessinger (Yale University). Constructs encoding myc-tagged Mnk1, Mnk1-T332D, and Mnk1-T2A2 were kindly provided by Jonathan Cooper (Fred Hutchinson Cancer Research Center). pUSE-Src was obtained from Upstate Biotechnology.

The following reagents at the indicated working concentrations were employed in our studies: 20 µM Mek inhibitor PD98059 (Promega), 10 µM p38 inhibitor SB203580 (Calbiochem), 20 µM Mnk inhibitor CGP57380 (kindly provided by Jerry Pelletier, McGill University, Canada), and 100 nM okadaic acid (Calbiochem).

Cell culture, transfection, and growth factor stimulation. CHO-K1 cells were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS) (Gibco). MIA PaCa-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 2.5% horse serum (Gibco). COS1 cells were maintained in DMEM supplemented with 5% FBS. All cells were cultured at 37°C in the presence of 5% CO2. CHO-K1 and COS1 cells were transfected using Fugene 6 reagent (Roche). After 1 day, the transfected cells were serum starved prior to growth factor stimulation (9, 18). For EGF stimulation, cells were incubated with ice-cold EGF stimulation medium (Ham's F-12 medium supplemented with 20 mM HEPES, pH 7.5, 0.1% bovine serum albumin [BSA], and 100 ng/ml recombinant human EGF [Invitrogen]) at 4°C for 15 min. Subsequently, the stimulation medium was replaced with 37°C prewarmed Ham's F-12 medium containing 20 mM HEPES and 0.1% BSA, and cells were incubated at 37°C for the indicated intervals. For FGF stimulation, cells were incubated with FGF stimulation medium (DMEM supplemented with 0.1% BSA, 500 ng/ml heparin, and 10 ng/ml bFGF [Invitrogen]) at 4°C for 15 min. Subsequently, the stimulation medium was replaced with 37°C prewarmed DMEM, and cells were incubated at 37°C for the indicated intervals.

Immunoprecipitation and immunoblot analysis. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in immunoprecipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP-40 alternative, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride [PMSF], 1 mM Na3VO4, 10 mM NaF, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM benzamidine). Cell lysates were passed through 26G3/8 needles three times and clarified by centrifugation at 12,000 x g for 10 min at 4°C. The clarified lysates were incubated with primary antibodies for 1 h at 4°C. Subsequently, the immune complexes were captured with protein A-Sepharose beads (Sigma) for 1 h at 4°C. The immunoprecipitated proteins were washed three times with ice-cold immunoprecipitation buffer and eluted from the beads by boiling in 2x sodium dodecyl sulfate (SDS) sample buffer.

Cell lysates or immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred onto nitrocellulose membranes. The primary antibodies used were anti-Spry2 polyclonal antibody (1:10,000), anti-HA tag 12CA5 monoclonal antibody (1:5,000), anti-myc tag antibody (1:1,000; Santa Cruz), anti-T7 tag monoclonal antibody (1:10,000; Novagen), anti-phosphotyrosine 4G10 monoclonal antibody (1:1,000; Upstate Biotechnology), antitubulin monoclonal antibody (1:20,000; Sigma), anti-ERK2 (1:1,000; Cell Signaling), and anti-phospho-ERK1/2 (1:1,000; Cell Signaling). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin Gs (1:10,000; Pierce).

Calf intestine phosphatase treatment. The Spry2 immunoprecipitates were first washed three times with immunoprecipitation buffer and then washed twice with phosphatase buffer (50 mM Tris, pH 8.8, 1 mM MgCl2, 0.1 mM ZnCl2, 1 mM PMSF). The Spry2 immunoprecipitates were then incubated for 30 min at 37°C in 100 µl of phosphate buffer containing 10 units of calf intestine alkaline phosphatase (Roche). Control incubations were performed in the absence of phosphatase. After washing three times with immunoprecipitation buffer, the immunoprecipitates were subjected to immunoblot analysis.

Protein expression. Two liters of XL1-Blue Escherichia coli cells harboring the pProExHTb constructs encoding hSpry21-172aa, hSpry2-S112A1-172aa, hSpry2-S121A1-172aa, or hSpry2-S112A/S121A1-172aa was grown to mid-log phase at 30°C. Protein expression was induced in the presence of 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 6 h. Bacterial cells were harvested and then lysed by sonication in resuspension buffer (PBS containing 20 mM HEPES, pH 7.4, 500 mM NaCl, ß-mercaptoethanol, 5 mM MgCl2, 20 mM imidazole, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM benzamidine, 1 mM PMSF, 10 µg/ml soybean trypsin inhibitor). The lysates were cleared by centrifugation for 15 min at 13,000 rpm and then incubated with 500 µl prewashed nickel-nitrilotriacetic acid beads on a rotator at 4°C. The beads were washed six times with resuspension buffer, and the proteins were eluted with 150 mM imidazole. The proteins were concentrated and washed in PBS containing 20 mM HEPES, pH 7.4, 500 mM NaCl, and 5 mM MgCl2 until the imidazole concentration was less than 1 mM. The samples were snap-frozen and stored at –80°C. Protein concentration and purity were determined by use of a Coomassie protein assay kit (Bio-Rad) and SDS-PAGE analysis followed by Coomassie staining.

Radiolabeling of hSpry2 and in vitro Mnk1 kinase assay. For in vivo radiolabeling of hSpry2, CHO-K1 cells transiently transfected with HA-hSpry2 were metabolically labeled with [32P]orthophosphate (1 mCi/ml; ICN) in phosphate- and serum-free DMEM for 4 h. The cells were subsequently stimulated with EGF for 10 min, and hSpry2 was immunoprecipitated with anti-HA antibodies.

For the in vitro Mnk1 kinase assay, CHO-K1 cells were transfected with myc-tagged Mnk1. Following EGF stimulation, myc-tagged Mnk1 was immunoprecipitated with anti-myc antibodies. The immunoprecipitated myc-Mnk1 was washed with immunoprecipitation buffer containing 0.5 M LiCl and incubated with purified hSpry2 proteins at 30°C for 30 min in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM ß-glycerophosphate, 25 µM ATP) containing 10 µCi [{gamma}-32P]ATP. Reaction mixtures were resolved by SDS-PAGE and analyzed by autoradiography.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serine phosphorylation of hSpry2 is associated with electrophoretic mobility shift. Ectopically expressed hSpry2 migrates on an SDS-polyacrylamide gel as three distinct bands with apparent molecular masses of 35, 42 and 45 kDa (Fig. 1A). In response to EGF stimulation, the relative abundance of the slowest-migrating form of hSpry2 increases significantly with a concomitant increase in 32P incorporation (Fig. 1A). These observations, along with the findings that hSpry2 immunoprecipitates treated with calf intestine alkaline phosphatase display only the most rapidly migrating form of the protein (Fig. 1A), indicate that the EGF-induced shift in the electrophoretic mobility of hSpry2 reflects its modification by phosphorylation. Spry proteins contain conserved tyrosine residues that become phosphorylated in response to growth factor stimulation (12, 18, 20, 34, 47, 50). In addition, phosphoamino acid analyses have indicated that Spry2 is serine phosphorylated (23; J. DaSilva and D. Bar-Sagi, unpublished data). The electrophoretic migration pattern of an hSpry2 mutant that is deficient in tyrosine phosphorylation is not altered from that of the wild-type hSpry2 (18, 47, 48), which suggests that the upward shift of hSpry2 arises as a consequence of serine phosphorylation. In support of this idea, we have observed that treatment of cells with okadaic acid (OA), a specific inhibitor of serine/threonine phosphatases, leads to the accumulation of the slow-migrating form of hSpry2 (Fig. 1B). This effect was observed under conditions previously described to specifically inhibit PP2A (10), raising the possibility that hSpry2 might be a substrate for PP2A. Indeed, the expression of a PP2A catalytic subunit resulted in the accumulation of the hypophosphorylated form of hSpry2 (Fig. 1C). Based on this analysis, we conclude that changes in the relative amounts of the slow- and fast-migrating forms of hSpry2 can be used to assess gain or loss, respectively, of serine phosphorylation. Similar phosphorylation-dependent changes in electrophoretic mobility patterns have been observed for endogenous Spry2 (Fig. 1D), indicating the in vivo relevance of Spry2 serine phosphorylation.


Figure 1
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  		FIG. 1. Growth factor-induced serine phosphorylation of hSpry2 results in reduced electrophoretic mobility of the protein. (A) CHO-K1 cells transfected with EGFR and HA-tagged hSpry2 were starved, metabolically labeled with [32P]orthophosphate, and subsequently treated with or without EGF as indicated. Cells were then lysed, and hSpry2 proteins were immunoprecipitated (IP) with anti-HA antibodies. The electrophoretic mobility of hSpry2 in the immunoprecipitates was examined by immunoblotting (IB) with anti-HA antibodies (left), and the incorporation of radioactivity was visualized by autoradiography (middle). hSpry2 immunoprecipitates were treated with calf intestine alkaline phosphatase (CIP), and immunoblot analysis (right) was conducted subsequently. (B) CHO-K1 cells transfected as described for panel A were serum starved and treated with vehicle or OA followed by EGF stimulation for 10 min. Cell lysates were prepared for immunoblot analysis. (C) Lysates from cells transfected with HA-hSpry2 and T7-PP2A-C{alpha} were analyzed by immunoblotting as indicated. (D) Cell lysates from serum-deprived MIA PaCa-2 cells treated with EGF, FGF, or OA as indicated were examined by immunoblotting for endogenous hSpry2. The data shown are representative of at least three independent experiments. Molecular masses (in kilodaltons) are shown to the left of the gels.

 
hSpry2 is a target of Mnk1. To gain insight into the identity of the kinase(s) that might mediate serine phosphorylation of hSpry2, we employed pharmacological agents that inhibit EGF-activated signaling pathways. Treatment of cells with the Mek inhibitor PD98059 interfered only partially with the EGF-induced accumulation of the hyperphosphorylated form of hSpry2 (Fig. 2). A similar effect was observed in the presence of the p38 MAPK pathway inhibitor SB203580 (Fig. 2) but not with the JNK inhibitor SP 600125 (data not shown). Significantly, the hSpry2 mobility shift was abrogated in the presence of both Mek and p38 MAPK inhibitors (Fig. 2), suggesting that the serine phosphorylation of hSpry2 is dependent on the concerted activity of both the ERK and p38 MAPK cascades. Consistent with this signaling profile, the constitutive activation of the ERK cascade by an oncogenic form of Ras (H-RasV12) or a membrane-targeted form of Raf (Raf-CAAX) resulted in the pronounced accumulation of the hyperphosphorylated form of hSpry2 (see Fig. S1 in the supplemental material).


Figure 2
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  		FIG. 2. EGF-induced serine phosphorylation of hSpry2 requires the concomitant activation of the Mek/ERK and p38 MAPK pathways. CHO-K1 cells transiently transfected with EGFR and HA-hSpry2 were incubated with the indicated MAPK pathway inhibitors for 6 h prior to EGF stimulation. Cells were lysed, and the electrophoretic mobility of hSpry2 was assessed by immunoblotting (IB). The data shown are representative of three independent experiments.

 
Both ERK and p38 MAPK have a specific requirement for a proline residue immediately following the phosphorylation site for efficient phosphorylation of their substrates (41). The lack of such a serine-containing consensus motif in the sequence of hSpry2 protein makes it unlikely that it serves as a direct substrate of these kinases. We thus turned our attention to kinases that are regulated by MAPKs, namely, the MAPK-activated protein kinases. Of the 11 kinases that comprise this family, i.e., Rsk1 to Rsk4, Msk1/2, MK2/3/5, and Mnk1/2 (45), in principle only Mnk1 fulfills the criteria of being the kinase for hSpry2 because it is a cytosolic kinase that depends on both ERK and p38 MAPK for its activation (45). Moreover, there are six serine-containing consensus sites for Mnk1 (K/RXXS, RXS [40, 45]) on hSpry2 at residues 7, 74, 112, 121, 125, and 167. To investigate the possible role of Mnk1 in the regulation of hSpry2 phosphorylation, we first examined whether hSpry2 is an in vitro substrate of Mnk1. As illustrated in Fig. 3A, a purified N-terminal fragment of hSpry2 containing all putative Mnk1 serine phosphorylation sites was phosphorylated by Mnk1 isolated from EGF-stimulated cells. Further site-directed mutagenesis studies in which all putative Mnk1 sites were substituted for alanine individually and collectively revealed that the sites phosphorylated by Mnk1 in vitro are Ser 112 and Ser 121 (Fig. 3A). Notably, only the faster-migrating form of hSpry2 was observed in cells expressing the hSpry2-S112A/S121A double mutant (Fig. 3B). Moreover, mutation of these sites abolished the ability of EGF to shift hSpry2 to its slower-migrating form and to induce an increase in phosphate content (Fig. 3B), indicating that hSpry2 might be a substrate of Mnk1 in vivo. To test this possibility, hSpry2 was expressed in the presence of constitutively active (T332D) or dominant interfering (T2A2) forms of Mnk1 (61). As illustrated in Fig. 3C, expression of activated Mnk1 led to a stoichiometric shift in the mobility of hSpry2 to the slower-migrating form both in the presence and in the absence of EGF. On the other hand, expression of the interfering Mnk1 mutant suppressed both the basal and EGF-stimulated hSpry2 migration shifts. Furthermore, expression of activated Mnk1 failed to induce the mobility shift of hSpry2 lacking Ser 112 and Ser 121 phosphorylation sites (Fig. 3C). Together, these results indicate that phosphorylation of hSpry2 on Ser 112 and Ser 121 in response to growth factor stimulation is mediated by Mnk1. This conclusion is supported by the observation that treatment of cells with the Mnk1-specific inhibitor CGP57380 (27) blocked the growth factor-induced phosphorylation of both ectopically expressed hSpry2 and endogenous Spry2 (Fig. 3D and E). The phosphorylation of hSpry2 by Mnk1 was not associated with the formation of a complex between the two proteins (data not shown).


Figure 3
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  		FIG. 3. Phosphorylation of hSpry2 on serines 112 and 121 is mediated by Mnk1. (A) CHO-K1 cells transfected with EGFR and myc-tagged Mnk1 were treated with EGF as indicated. Mnk1 immunoprecipitated from the cell lysates was incubated with [{gamma}-32P]ATP and the indicated N-terminal fragments of hSpry2 (hSpry21-172aa) that were purified from bacteria. The hSpry21-172aa peptides were subjected to SDS-PAGE and stained with Coomassie blue (Coomassie), and the incorporation of radioactivity was determined by autoradiography. (B) CHO-K1 cells transfected with EGFR and the indicated hSpry2 proteins were labeled with [32P]orthophosphate and analyzed as described for Fig. 1A. (C) CHO-K1 cells expressing EGFR and indicated proteins were serum starved and treated with EGF, and the electrophoretic mobility of hSpry2 was assessed by immunoblotting. (D) CHO-K1 cells transfected with EGFR and hSpry2 were serum starved and incubated with CGP57380 for 16 h prior to EGF stimulation and immunoblot analysis. (E) MIA PaCa-2 cells were serum deprived and treated with CGP57380 for 16 h prior to EGF stimulation for 15 min, and the lysates were examined by immunoblot analysis for endogenous hSpry2 and tubulin. The data shown are representative of at least three independent experiments. IB, immunoblotting.

 
Serine phosphorylation of hSpry2 protects it against degradation. It has been previously shown that growth factor stimulation promotes the degradation of hSpry2 through the proteasomal pathway (18, 47). To examine the effects of hSpry2 serine phosphorylation on this process, we have compared the rates of EGF-induced degradation of wild-type and S112A/S121A double mutant forms of hSpry2. As illustrated in Fig. 4A, the decrease in steady-state levels of hSpry2-S112A/S121A following EGF stimulation occurred appreciably faster than that for the wild-type protein. Indeed, whereas a significant fraction of the starting level of wild-type hSpry2 was still present 15 min after growth factor stimulation, only trace amounts of the S112A/S121A mutant were observed at this time point. Since the accelerated decrease in the levels of S112A/S121A was blocked by the addition of the proteasome inhibitor MG132 (Fig. 4A), these observations suggest that serine phosphorylation is required to protect hSpry2 from proteolytic degradation. Accordingly, conditions that enhance hSpry2 serine phosphorylation would be predicted to attenuate its proteolytic degradation, and conversely, conditions that prevent hSpry2 serine phosphorylation would be predicted to accelerate hSpry2 proteolytic degradation. Consistent with these predictions, the hyperphosphorylated form of hSpry2 generated by the treatment of cells with OA was resistant to EGF-induced degradation, and the hypophosphorylated form of hSpry2 generated as a consequence of inhibiting the p38 and ERK cascades displayed a high degradation rate compared to the wild-type protein (Fig. 4B). In addition, the expression of constitutively active Mnk1 slowed down the EGF-induced degradation of hSpry2, whereas inhibition of Mnk1 activity by either dominant interfering Mnk1 or Mnk1 inhibitor CGP57380 resulted in the enhanced degradation of both ectopically expressed and endogenous hSpry2 (Fig. 4B and C and 3E). Therefore, the serine phosphorylation-dependent stabilization of hSpry2 following EGF stimulation appears to be mediated by Mnk1. A similar role for serine phosphorylation was observed in the context of FGF stimulation (see Fig. S2A in the supplemental material), suggesting that the link between hSpry2 serine phosphorylation and metabolic stabilization might be a general regulatory feature of the hSpry2 feedback loop.


Figure 4
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  		FIG. 4. Growth factor-induced serine phosphorylation of hSpry2 protects it against degradation. (A) CHO-K1 cells were transfected with EGFR and the indicated hSpry2 proteins. The cells were serum starved, treated with or without MG132, and stimulated with EGF for the indicated intervals. The levels of hSpry2 were assayed by immunoblotting (IB) with anti-HA antibodies (top), and equal loading of samples was confirmed by immunoblotting with antitubulin antibodies (bottom). (B) CHO-K1 cells expressing hSpry2 were treated with indicated inhibitors, followed by EGF stimulation and immunoblot analysis as described for panel A. (C) CHO-K1 cells were transfected with EGFR and hSpry2 along with constitutively active Mnk1 (Mnk1-T332D) or dominant interfering Mnk1 (Mnk1-T2A2). The cells were subsequently stimulated with EGF for the indicated intervals, and the lysates were analyzed as described for panel A. Mnk1 was detected by immunoblotting with anti-myc antibodies.

 
Serine phosphorylation of hSpry2 negatively regulates tyrosine 55 phosphorylation and c-Cbl binding. The targeting of hSpry2 for proteolytic degradation depends on the phosphorylation of a conserved tyrosine residue at position 55 (12, 18, 47). This phosphorylation event promotes the binding of hSpry2 to the E3 ubiquitin ligase c-Cbl, which results in the ubiquitination and subsequent degradation of the protein (12, 18, 47). To test whether this mechanism is responsible for the enhanced proteolytic degradation displayed by hSpry2-S112A/S121A, we generated an hSpry2 mutant lacking both the serine and tyrosine phosphorylation sites (Y55F/S112A/S121A). In agreement with previous results (18), hSpry2 does not undergo degradation if phosphorylation of Tyr 55 is prevented (Fig. 5A). Likewise, functional ablation of Tyr 55 was sufficient to block the degradation of the serine phosphorylation-deficient mutant of hSpry2 (Fig. 5A). Similar results were obtained when the stability of these hSpry2 mutants was examined following FGF stimulation (see Fig. S2B in the supplemental material). These results indicate that the accelerated degradation of hSpry2 observed in the absence of serine phosphorylation occurs as a consequence of alterations in the pathway that couples Tyr 55 phosphorylation to hSpry2 degradation.


Figure 5
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  		FIG. 5. Serine phosphorylation of hSpry2 interferes with Tyr 55 phosphorylation and c-Cbl binding. (A) Cell lysates prepared from CHO-K1 cells transfected with EGFR and indicated vectors were analyzed as described in the legend to Fig. 4A. (B) CHO-K1 cells were transfected with EGFR, HA-tagged c-Cbl, and the indicated hSpry2 expression plasmids. The cells were subsequently serum deprived and treated with or without EGF for 5 min. hSpry2 was immunoprecipitated with anti-Spry2 antibodies, and the presence of c-Cbl in the immunocomplexes was determined by immunoblotting. (C) hSpry2 immunoprecipitates were isolated from serum-deprived or EGF-stimulated CHO-K1 cells expressing EGFR and indicated hSpry2 proteins, and tyrosine phosphorylation (pTyr) was determined by immunoblotting. (D) CHO-K1 cells expressing the indicated proteins were serum starved prior to lysis, and the hSpry2 immunoprecipitates were analyzed as described for panel C. Note that in the experiments described in panels B, C, and D, amounts of transfected plasmids were adjusted to achieve comparable levels of protein expression for the wild type and for the S112A/S121A mutant of hSpry2. The data shown are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation.

 
As pointed out above, the principal role of Tyr 55 phosphorylation in the context of hSpry2 proteolytic degradation is to provide a docking site for c-Cbl. To determine the effects of serine phosphorylation on the interaction of hSpry2 with c-Cbl, cells were cotransfected with c-Cbl and various hSpry2 constructs and complex formation was assessed by coimmunoprecipitation assays. As illustrated in Fig. 5B, the EGF-dependent binding of c-Cbl to hSpry2-S112A/S121A was augmented relative to that of the wild-type protein. The interaction of hSpry2-S112A/S121A with c-Cbl was strictly dependent on Tyr 55 phosphorylation, as is evident from the observation that the hSpry2-Y55F/S112A/S121A mutant lost its ability to interact with c-Cbl (Fig. 5B). To test whether the inverse correlation between serine phosphorylation of hSpry2 and c-Cbl binding is a consequence of changes in the status of Tyr 55 phosphorylation, we compared the extents of Tyr 55 phosphorylation in wild-type and S112A/S121A mutant forms of hSpry2. As shown in Fig. 5C, the capacity of hSpry2 to undergo EGF-induced tyrosine phosphorylation on Tyr 55 was significantly enhanced in the absence of serine phosphorylation. Similar effects were observed following FGF stimulation (see Fig. S3 in the supplemental material). Since Tyr 55 of hSpry2 has been shown to be the target of the Src family of kinases (30, 34), we next asked whether hSpry2-S112A/S121A is a preferential substrate for Src. Figure 5D demonstrates that ectopically expressed Src induced the tyrosine phosphorylation of hSpry2-S112A/S121A to a much greater extent than wild-type hSpry2; this effect was not observed when Tyr 55 was mutated. Together, these results demonstrate that in response to growth factors, serine phosphorylation of hSpry2 stabilizes the protein by interfering with its Tyr 55 phosphorylation and binding to c-Cbl.

Serine phosphorylation modulates the inhibitory effect of hSpry2 on FGF signaling. To investigate the functional significance of hSpry2 serine phosphorylation, we compared the effects of wild-type and hSpry2-S112A/S121A on FGF-induced ERK activation. In agreement with previous reports (7, 15, 18, 20), the expression of wild-type Spry resulted in the attenuation of the delayed phase of FGF-induced ERK activation (Fig. 6). In contrast, the profile of ERK activation in response to FGF was not altered by the expression of hSpry2-S112A/S121A (Fig. 6). Since the degradation rate of this mutant is considerably greater than that of the wild-type protein, it is likely that the loss of the inhibitory effect is a consequence of the reduction in the amounts of the mutant to a level that is below a critical threshold. These results underscore the importance of the tight regulation of the amounts of hSpry2 for its inhibitory activity and implicate serine phosphorylation as a major element of the circuitry that contributes to this regulation.


Figure 6
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  		FIG. 6. Serine phosphorylation modulates the inhibitory effect of hSpry2 on FGF signaling. COS1 cells transfected with c-Cbl, HA-tagged ERK2, and the indicated hSpry2 vectors were serum deprived and treated with FGF for the indicated intervals. HA-ERK2 proteins were immunoprecipitated (IP) with anti-HA antibodies and subsequently immunoblotted (IB) with anti-ERK2 and anti-pERK antibodies. hSpry2 protein levels in the lysates were examined by immunoblotting with anti-Spry2 antibodies. The data shown are representative of three independent experiments.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RTK feedback inhibitors critically influence cell signaling decisions by modulating the strength and duration of downstream signals. In this report, we demonstrate that the conserved RTK inhibitor Spry2 is a substrate of the ERK/p38-dependent serine kinase Mnk1. Phosphorylation of hSpry2 on Ser 112 and Ser 121 by Mnk1 antagonizes the phosphorylation of hSpry2 on Tyr 55. Consequently, serine phosphorylation of hSpry2 stabilizes the protein by interfering with tyrosine-dependent degradation via the ubiquitin-proteasome pathway. These findings indicate that hSpry2 accumulation can be adjusted posttranslationally through the combined effect of multiple phosphorylation events and provide new insights into the involvement of Mnk1 in the regulation of RTK pathways.

Phosphorylation-induced alteration in the metabolic stability of proteins is a widely used mechanism by which protein levels can be modulated rapidly and reversibly. The protective role that serine phosphorylation plays with respect to proteolytic degradation is not unprecedented. For example, phosphorylation on Ser 362 and Ser 374 of the proto-oncoprotein c-Fos by c-Mos/Mek/ERK pathway stabilizes c-Fos and augments its transforming activity in NIH 3T3 cells (36); phosphorylation on Ser 322, Ser 326, and Ser 330 of the Wnt signaling inhibitor axin by glycogen synthase kinase-3ß stabilizes axin (64); and phosphorylation on Ser 146 of the cyclin-dependent kinase inhibitor p21Cip1 by AKT/protein kinase B stabilizes the protein and promotes survival (31).

The regulated targeting of Spry to proteolytic degradation is a multistep process, involving ligand-dependent Tyr 55 phosphorylation and the subsequent binding of the E3 ubiquitin ligase c-Cbl (12, 18, 34, 47, 63). As a consequence of this binding event, Spry undergoes polyubiquitination followed by degradation. We have found that serine phosphorylation interferes with the first step in the proteolytic cascade of Spry, namely, its tyrosine phosphorylation. It has been shown that Tyr 55 phosphorylation is mediated by Src-related kinases (30, 34). Upon serine phosphorylation, Spry becomes a poor substrate for Src, indicating that this modification restricts the accessibility of Spry to the modifying tyrosine kinase. Since serine phosphorylation of Spry does not appear to alter significantly its subcellular distribution (data not shown), it is unlikely that the lack of productive interactions between serine-phosphorylated Spry and the tyrosine kinase reflects changes in the spatial relationships between the two proteins. Rather, it is plausible that the serine phosphorylation of Spry induces a conformational change that hinders the interactions between Spry and the tyrosine kinase. Further biochemical studies will be required to test this hypothesis. Although in this study we have focused on the reciprocal relationship between serine and tyrosine phosphorylation in the context of the metabolic stability of Spry, it should be noted that under some circumstances Tyr 55 has been also implicated in mediating some of the inhibitory effects of Spry on RTK/ERK signaling (19, 20, 34, 50). Hence, it will be of importance to determine the relationship between serine phosphorylation and the antagonistic function of Spry.

To date, three substrates of Mnk1 have been identified: eukaryotic initiation factor 4E (eIF4E), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and cytosolic phospholipase A2 (cPLA2). These are involved in the regulation of 5' cap-dependent mRNA translation, 3' untranslated region-responsive translational repression, and arachidonate-derived signaling, respectively (4, 21, 60, 61). Our finding that hSpry2 is a substrate of Mnk1 identifies a novel link between this kinase and the regulation of ERK signaling. In mammalian cells, Mnk1 is activated by ERK and p38 in response to growth factors, cytokines, and cellular stress (14, 45, 60). The closest functional ortholog of Mnk family kinases in Drosophila is Lk6 kinase (2, 39). Lk6 has initially been described as a microtubule- and centrosome-associating protein (25), and recent studies have demonstrated a diet-dependent role for Lk6 in the regulation of cell growth (44). In addition, and most relevant to our findings, Lk6 has been identified in a modifier screen as a putative negative regulator of Ras/ERK signaling in Drosophila eye development (22). Specifically, overexpression of Lk6 in the background of constitutively active Ras partially suppresses the rough eye phenotype resulting from the activation of Ras pathway (22). The overexpression of dSpry produces a similar effect, and dSpry has been shown to be an essential negative regulator of EGF receptor signaling during Drosophila eye development (5, 28, 55). Our observation that Mnk1 promotes the stabilization of Spry provides a possible mechanistic explanation for the apparent similarities between the genetic interactions mediated by Lk6 and dSpry. Analogous to the effects of Mnk1 on Spry levels in mammalian cells, the phosphorylation of dSpry by Lk6 would be predicted to contribute to its antagonistic function by prolonging its metabolic half-life. The presence of Mnk1 consensus sites in dSpry as well as in all mammalian Spry proteins lends support to this idea and suggests that Mnk1-mediated Spry phosphorylation represents a conserved signaling mechanism to prolong the duration of Spry activity.

The serine phosphorylation state of hSpry2 not only is positively regulated by the action of the serine kinase Mnk1 but also appears to be reversed by the action of serine phosphatases. We provide evidence that the latter could be accomplished, at least in part, by PP2A, a ubiquitous serine phosphatase that has been implicated in the control of many phosphorylation-dependent regulatory pathways (24). Significantly, biochemical and genetic studies have established that PP2A can positively regulate Ras/ERK signaling (54, 62). Although the precise mechanism by which this positive regulation is accomplished is not completely understood, a recent study has demonstrated that PP2A mediates the assembly of active ERK signaling by dephosphorylating KSR and Raf and promoting their translocation to the plasma membrane (37). Given the inverse correlation we have observed between the serine phosphorylation and the metabolic stability of Spry, the action of PP2A would be predicted to reduce the levels of Spry by enhancing its proteolytic degradation. Thus, our data suggest that PP2A may positively influence the signaling output of the Ras/ERK pathway by removing negative feedback inhibition.

Changes in the duration and/or intensity of ERK signaling can lead to profound alterations in the biological outcome. Because Spry functions as a negative regulator of the RTK/ERK pathway, its levels need to be precisely controlled in order to ensure that the physiological response is correctly specified. In this study, we describe a mechanism that promotes the accumulation of Spry by delaying its proteolytic degradation. This mechanism involves the Mnk1-dependent phosphorylation of Spry on conserved serine residues. Since Mnk1 activity is regulated by ERK, the stabilization of Spry by serine phosphorylation provides a mechanism by which the duration of Spry activity can be rapidly modified in a manner dependent on the strength of ERK signals (Fig. 7). A remarkably similar scenario has been described for the MAP kinase inhibitor MKP-1. Like Spry, MKP-1 is transcriptionally induced by RTK signaling and thus is considered to be a component of a negative feedback loop that regulates ERK activity (56). Furthermore, MKP-1 is subject to proteasome-mediated degradation which is antagonized by ERK-dependent serine phosphorylation (3). Thus, it appears that the posttranslational control of protein concentration may have been selected as an effective means to determine the temporal range of antagonists of RTK/ERK signaling. Future studies aimed at achieving a quantitative understanding of the relationships between the different mechanisms that regulate Spry levels should provide insights into the determinants that govern the output capacity of RTK signaling.


Figure 7
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  		FIG. 7. A schematic representation of RTK-dependent signals that control the accumulation of hSpry2. The levels of hSpry2 reflect the balance between three signaling activities: ERK-mediated transcriptional upregulation, Mnk1-mediated serine phosphorylation, and Src-mediated tyrosine phosphorylation.

 


   ACKNOWLEDGMENTS
 
We thank Jerry Pelletier, Jonathan Cooper, David Brautigan, Joseph Schlessinger, and Daniel DiMaio for reagents and cDNA constructs. We also thank all members of the laboratory for helpful advice and discussions and James Keller for the generation of anti-Spry2 antibodies.

This work was supported by National Institutes of Health grant CA28146 and New York State Empire grant CO17941 (D.B.-S.) and by a Department of Defense predoctoral fellowship (H.J.K.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794. Phone: (631) 632-9737. Fax: (631) 632-5782. E-mail: barsagi{at}pharm.sunysb.edu. Back

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

{dagger} J.D. and L.X. contributed equally to this work. Back


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Molecular and Cellular Biology, March 2006, p. 1898-1907, Vol. 26, No. 5
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