A Back-Door Insights into the modulation of Src kinase activity by the polyamine spermidine

Src is a protein tyrosine kinase commonly activated downstream of transmembrane receptors and plays key roles in cell growth, migration and survival signaling pathways. In conventional dendritic cells (cDCs), Src is involved in the activation of the non-enzymatic functions of indoleamine 2,3-dioxygenase 1 (IDO1), an immunoregulatory molecule endowed with both catalytic activity and signal transducing properties. Prompted by the discovery that the metabolite spermidine confers a tolerogenic phenotype on cDCs that is dependent on both the expression of IDO1 and the activity of Src kinase, we here investigated the spermidine mode of action. We found that spermidine directly binds Src in a previously unknown allosteric site located on the backside of the SH2 domain and thus acts as a positive allosteric modulator of the enzyme. Besides confirming that Src phosphorylates IDO1, here we showed that spermidine promotes the protein-protein interaction of Src with IDO1. Overall, this study may pave the way toward the design of allosteric modulators able to switch on/off the Src-mediated pathways, including those involving the immunoregulatory protein IDO1.


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Besides being intermediates in metabolic reactions, metabolites can serve as intracellular and 25 intercellular signals (1). Indeed, by interacting with specific molecular partners, soluble mediators can 26 trigger a series of molecular events critical for cell fitness and adaptation. Metabolites binding to either 27 the active site or the allosteric pocketi.e., that different from the catalytic siteof enzymes are among 28 the best-characterized interactions that modulate protein activity as well as the assembly and function of 29 multiprotein complexes (2-4). 30 The naturally occurring polyamines (i.e., putrescine, spermidine and spermine) are organic cations 31 derived from the decarboxylation of L-ornithine, which is generated by the arginase 1 from L-arginine 32 (5,6). The conversion of L-ornithine into putrescine is catalyzed by the rate-limiting enzyme ornithine 33 decarboxylase 1, followed by two specific synthases that sequentially give rise to spermidine and 34 spermine (7). These metabolites are protonated at physiological pH levels, allowing them to interact with 35 negatively charged macromolecules, including nucleic acids, proteins, and phospholipids. Given their 36 structure, polyamines indeed modulate several cellular processes, ranging from cell growth and 37 proliferation to immune system function (8,9). As a matter of the fact, alteration of polyamines 38 intracellular content is associated with the occurrence of several tumors, including prostate, breast, and 39 colon cancers, for which polyamines are considered as biomarkers (10-12). Among polyamines, 40 spermidine has recently gained much more attention as player of immune regulation and in age-related 41 disorders, such as cardiac hypertrophy and memory impairment (13-18). Spermidine exerts a protective 42 role in mouse experimental models of autoimmune diseases, such as multiple sclerosis and psoriasis, by 43 activating the Forkhead box protein O3 (FOXO3) pathway and thus suppressing the production of 44 inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 (14). Moreover, spermidine 45 is able to reprogram mouse conventional dendritic cells (cDCs) toward an immunoregulatory phenotype 46 via Src kinase-dependent phosphorylation of indoleamine 2,3-dioxygenase 1 (IDO1) (19). However, the 47 exact mechanism of Src activation by spermidine remains to be elucidated. 48 The non-receptor tyrosine kinase Src is the representative of a family of structure-related kinases initially 49 discovered as a proto-oncogene regulating critical cellular functions (20). Src activation mainly occurs 50 downstream of multiple transmembrane receptors, including epidermal growth factor receptor (EGF-R), 51 fibroblast growth factor receptor (FGF-R), and insulin-like growth factor-1 receptor (IGF-1R). Indeed, a 52 dysregulated Src activity has been associated with tumor growth and metastasis, inflammation-mediated 53 carcinogenesis, and therapeutic resistance to traditional antineoplastic drugs (21-26). The induction of 54 Src kinase activity can also occur following Aryl hydrocarbon Receptor (AhR) activation, whose 55 conformational changes favor the Src-AhR disjunction, allowing the former to phosphorylate its 56 downstream partner IDO1 and thereby promote the generation of an immunoregulatory milieu (27). 57 In addition to the kinase domain, Src possesses an N-terminal Src homology-4 (SH4) domain, a unique 58 domain, an SH3 domain, an SH2 domain, an SH2-kinase linker domain, and a C-terminal autoregulatory 59 motif (25). The SH2 and SH3 modules serve in protein-protein interactions that are essential for the 60 regulation of kinase activity and signaling function. Specifically, the SH2 domain contains two distinct 61 binding pockets. The first one has a conserved arginine residue that binds a phosphotyrosine (pY) residue 62 presented by the protein substrate, whereas the second pocket binds the residue that is three positions C-63 terminal of pY (pY+3), contributing to the specificity in ligand protein recognition. The autoregulatory function of the kinase occurs through intramolecular interactions that stabilize the 65 catalytically inactive conformation of Src, in which the SH2 domain binds to a pY located at position 66 +535. Accordingly, binding of ligand proteins to the SH2 domain displaces intramolecular contacts and 67 promotes the catalytic activation of the Src kinase, which is characterized by the phosphorylation of a 68 tyrosine residue in the activation loop (Y424). Given the crucial role of the non-catalytic domains in 69 modulating Src kinases activity, efforts have been made to develop drug-like modulators of the SH2 and 70 SH3 domains. Small peptidomimetics destabilize the closed conformation and thus promote the kinase 71 activation through the binding of SH3 and/or SH2 domains (28,29). Alternatively, modulators of Src 72 kinases able to reinforce the intramolecular interactions have proven to allosterically inhibit the enzyme 73 activity (30).

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Prompted by the finding that spermidine triggers the immunosuppressive IDO1 signaling in cDCs (19), 75 here we investigated the molecular relationship between that polyamine, Src kinase and IDO1. We found 76 that spermidine (i) activates Src kinase with an allosteric mechanism; (ii) binds directly Src kinase at a 77 previously unknown allosteric site; (iii) favors the association of IDO1 and Src kinase.

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Spermidine causes allosteric activation of the kinase activity of Src 80 The activation of Src kinase mainly occurs downstream of multiple transmembrane and intracellular 81 receptors (such as AhR) as well as protein tyrosine phosphatases (27,31,32). In cDCs, it has been 82 demonstrated that a small molecule, namely spermidine, activates Src with a still undefined mechanism 83 (19). To figure out whether a direct activation would occur, we assayed spermidine against purified 84 recombinant human Src (rhSrc) protein. After 30 minutes of incubation, a luminescent assay was used to 85 measure the ADP released by the kinase. Results showed that spermidine activated rhSrc with a half-86 maximal effective concentration (EC50) of 106.4 nM ± 13.4 (Fig 1A). To confirm the modulation of the 87 kinase also in living cells, we resorted to immunoblot analysis of phosphorylated Src at the tyrosine Y424 88 as sign of kinase activation. SYF cells (i.e., fibroblast null for Src family kinases, Src, Yes, and Fyn (33)) 89 were stably transfected with vector encoding for Src kinase and then treated with increasing 90 concentration of spermidine. Results showed that the metabolite promoted Src phosphorylation with an 91 EC50 of 6.4 μM ± 0.6 (Fig 1B, 1C). 92 To get insights into the mechanism of action of spermidine, we measured the intrinsic activity of the 93 polyamine in the absence of either ATP or the synthetic peptide. Results showed that spermidine did not 94 activate Src in the absence of either ATP or peptide (Fig 1D), while it promoted the production of ADP 95 when the substrate is also present, ruling out any competition for the same site. As this profile was 96 compatible with an allosteric modulation, we incubated rhSrc with different concentration of ATP or 97 peptide. In the presence of fixed amount of spermidine and increasing concentration of the peptide, the 98 maximum rate of Src kinase activity (Vmax) increased, while the affinity (Km) for the substrate was not 99 affected (Fig 1E). On the contrary, in the presence of different concentration of ATP, spermidine did not 100 modify neither the efficacy nor the affinity of Src kinase (Supplementary Fig S1). Such a kinetic profile 101 is consistent with a non-ATP competition, suggesting that spermidine allosterically activates the kinase 102 activity of Src. In the inactive state, Src assumes a closed conformation with the SH3 domain bound to the SH2-kinase 105 linker and the SH2 domain bound to the tyrosine phosphorylated tail (Figure 2A). Using the 106 experimental available structure of SH2 domain (PDB ID: 2JYQ) and electrostatic potential calculations, 107 we characterized key structural and electrostatic elements of the SH2 domain involved in ligand protein 108 recognition (Fig 2B). A positive electrostatic potential was observed in the region of the pY binding site 109 (R183 and H209 residues, Fig 2B), whereas a stretch of surface endowed with a strong negative 110 electrostatic potential was observed on the backside of the pY binding site as delimited by glutamate 111 residues E155 and E174 (Fig 2B), suggesting the existence of a putative allosteric site. Of note, by the 112 alignment of amino acid sequences, we identified that such residues were conserved in Src protein of 113 human, murine, chicken and rat (Supplementary Fig S2), further supporting potential functional role 114 for this allosteric site.  Table S1; Fig 2D), the first primary amine group interacts by an 119 electrostatic enforced hydrogen bond with E155, the secondary amine group forms electrostatic enforced 120 hydrogen bond with E155 and the carbonyl group of T255, the other primary amine group makes 121 hydrogen bonds with the side chain of E155 and the carbonyl group of A153 while engaging the aromatic 122 ring of F155 through a specific π-cation interaction (34).

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To experimentally confirm the proposed spermidine binding site, we resorted to mutagenesis experiments 124 by substituting the glutamate residues 155 or 174 of murine Src into alanine (E155A; E174A). SYF cells 125 were thus stably transfected with vectors coding for the mutated Src (i.e., Src E155A and Src E174A) 126 and wild-type Src (WT) (Supplementary Fig S3A). To validate the functional equivalence of Src 127 mutants, cells were exposed to lysophosphatidic acid (LPA), a stimulus known to activate Src kinase  Fig S3B). On evaluating the activation of Src by spermidine, we found that the 131 mutation of the glutamate residues abrogated the kinase activation (Fig 2E, 2F). The split-luciferase 132 fragment complementation assay confirmed that E155 and E174 are key anchoring points for spermidine 133 binding. Specifically, SYF cells expressing Src WT or mutant were stably transfected with a 134 bioluminescent reporter that contains the SH2 domain and the Src consensus substrate peptide between 135 the amino-(Nluc) and carboxyl-(Cluc) terminal domains of the Firefly luciferase molecule (Fig 2G) (36).

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When the endogenous Src is active, the tyrosine residue of the consensus peptide is phosphorylated and 137 interact with the docking pocket of the SH2 domain. This creates a steric hindrance that prevents the 138 reconstitution of a functional luciferase, resulting in a reduction of bioluminescent signal (Fig 2G). Cells 139 co-expressing Src and the reporter were thus exposed to spermidine and the luminescent signal was 140 measured. Results demonstrated that the bioluminescence decreased when spermidine is applied only in 141 cells ectopically expressing wild-type Src (Fig 2H). 142 Overall, these data suggested the presence of a previously unknown allosteric site on the backside of Src 143 SH2 domain as defined by the glutamate residues at position 155 and 174. Spermidine, by means of ionic 144 and hydrogen bond interactions between its protonated amino groups and residues of the shallow anionic 145 site on the SH2 domain, directly associates with and activates Src kinase. It is worth noting that no direct 146 6 interaction was observed between spermidine and E174 in the docking study. This may be ascribed to 147 the limit of the scoring function in identifying a binding mode engaging E174 among resulting solutions, 148 or to an indirect role of such residue in promoting long range electrostatic interactions to accomplish the 149 molecular recognition of the cognate ligand into the allosteric site.

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Spermidine promotes the Src-dependent tyrosine phosphorylation of IDO1 and their interaction 151 Among the proteins phosphorylated by Src, the immunometabolic enzyme IDO1 is worthy of note 152 (19,27). Indeed, aside metabolizing the amino acid tryptophan, IDO1 is endowed with non-enzymatic 153 properties (31,37-39). The latter relies on the presence of two ITIMs (immunoreceptor-tyrosine based 154 inhibitory motif) that can be phosphorylated in response to immunomodulatory stimuli, such as TGF-β, 155 L-kynurenine and spermidine (19,27,38). However, the exact molecular mechanism and the role of 156 spermidine have never been explored. To confirm that Src can phosphorylate IDO1, SYF cells were 157 reconstituted with vectors coding for wild-type Src and IDO1, either alone or in combination, and then 158 were exposed to spermidine. Results from immunoblot demonstrated that the co-precipitated IDO1 is 159 tyrosine phosphorylated by Src and that the polyamine increases the phosphorylation (Fig 3A). To further 160 confirm that spermidine could promote the IDO1 phosphorylation by accelerating the reaction velocity, 161 an in vitro kinase assay was performed using purified Src and IDO1 protein. By detecting 162 phosphotyrosine residues with a specific antibody, we found that IDO1 was phosphorylated in a time 163 dependent manner (Fig 3B, 3C). Moreover, in the presence of spermidine, Src quicker phosphorylated 164 IDO1, as demonstrated by the 2-fold increase of the relative velocity (Fig 3D). To figure out whether the 165 IDO1 phosphorylation was a direct effect through physical interaction with Src, SYF cells reconstituted 166 with wild-type Src and IDO1 were exposed to spermidine for different length of time. Co-167 immunoprecipitation followed by immunoblot studies demonstrated that when cells were treated with 168 spermidine for 60 minutes, IDO1 was found in a complex with Src (Fig 3E). The specific IDO1-Src 169 interaction was confirmed in situ by the proximity ligation assay (Fig 3F, 3G). Accordingly, spermidine 170 treatment induced the Src-IDO1 interaction in SYF cells reconstituted with wild-type Src, but not with 171 the E155A or E174A mutant form of the kinase (Fig 3F, 3G). 172 As a whole, these results suggested that spermidine not only accelerates the Src-mediated 173 phosphorylation of IDO1, but also promotes the formation of Src-IDO1 complex. inhibitors (TCIs)has been pursued at the preclinical level for blocking Src kinase activity (43). 188 However, the promiscuity of molecules interacting with the ATP pocket has moved the interest toward 189 the development of alternative strategies for more effective and less-toxic inhibitors.

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The peculiarity of the Src protein, as well as of other tyrosine kinases, is its structural plasticity, i.e., the 191 capability to adopt distinct conformations due to intrinsic dynamic properties (44). The activation state  Besides confirming that Src phosphorylates IDO1, and that the polyamine accelerates the enzyme kinetic, 229 here we showed that spermidine promotes the interaction of Src with IDO1 protein (Fig 4). Our data 230 provided evidence that an endogenous metabolite, when present at specific concentrations, can directly                Table S1). E155 and E174 residues are shown with magenta carbon-  Table S1). E155 and E174 are shown 389 with magenta carbon-atoms. Interacting residues and spermidine are shown with grey and green carbon-   or Src mutated at glutamate 155 or 174 with alanine and then exposed to LPA (20 μM).