Neutralising properties of peptides derived from CXCR4 extracellular loops towards CXCL12 binding and HIV-1 infection

The chemokine receptor CXCR4 interacts with a single endogenous chemokine, CXCL12, and regulates a wide variety of physiological and pathological processes including inflammation and metastasis development. CXCR4 also binds the HIV-1 envelope glycoprotein, gp120, resulting in viral entry into host cells. Therefore, CXCR4 and its ligands represent valuable drug targets. In this study, we investigated the inhibitory properties of synthetic peptides derived from CXCR4 extracellular loops (ECL1-X4, ECL2-X4 and ECL3-X4) towards HIV-1 infection and CXCL12-mediated receptor activation. Among these peptides, ECL1-X4 displayed anti-HIV-1 activity against X4, R5/X4 and R5 viruses (IC₅₀ = 24 to 76 μM) in cell viability assay without impairing physiological CXCR4–CXCL12 signalling. In contrast, ECL2-X4 only inhibited X4 and R5/X4 strains, interfering with HIV-entry into cells. At the same time, ECL2-X4 strongly and specifically interacted with CXCL12, blocking its binding to CXCR4 and its second receptor, CXCR7 (IC₅₀ = 20 and 100 μM). Further analysis using mutated and truncated peptides showed that ECL2 of CXCR4 forms multiple contacts with the gp120 protein and the N-terminus of CXCL12. Chemokine neutralisation was mainly driven by four aspartates and the C-terminal residues of ECL2-X4. These results demonstrate that ECL2 represents an important structural determinant in CXCR4 activation. We identified the putative site for the binding of CXCL12 N-terminus and provided new structural elements to explain the recognition of gp120 and dimeric CXCR4 ligands.     

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.     

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface,Promoting Oligomerization,and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.     

Small neutralizing molecules to inhibit actions of the chemokine CXCL12

The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.     

CXCL14 is no direct modulator of CXCR4

C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.     

Design,synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]_n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL12_1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG₃]₂ linked homodimeric DV3, was an effective CXCR4 antagonist (IC₅₀ = 22 nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3–CXCL12_1–8, was an effective CXCR4 agonist (IC₅₀ = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.     

C-Terminal Engineering of CXCL12 and CCL5 Chemokines: Functional Characterization by Electrophysiological Recordings

Chemokines are chemotactic cytokines comprised of 70–100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K⁺ channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.     

CXCR4/CXCL12 在肿瘤中的研究进展

研究表明趋化因子及其受体在胚胎发育、干细胞迁移以及各种免疫反应中发挥重要作用,是许多生理及病理过程中细胞运动的重要因素。趋化因子受体CXCR4是一个由352个氨基酸构成的、7次跨膜的G蛋白偶联受体。趋化因子CXCL12为其特异性受体。研究发现,CXCR4/CXCL12在多种肿瘤中都有表达,在肿瘤的生长、血管生成、转移等方面发挥着重要作用。与正常组织相比,肿瘤组织及转移灶CXCR4高表达。因此,对CXCR4/CXCL12轴在肿瘤病生理中的作用机制进行进一步研究,很可能为肿瘤的治疗及对肿瘤转移的预防提供一个新的思路。我们现在就对其在肿瘤病生理中的作用做一综述。     

Structural and functional basis of CXCL12 (stromal cell-derived factor-1 alpha) binding to heparin

CXCL12 (SDF-1alpha) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional (1)H-(15)N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to beta-strands in the dimer interface. The second includes the amino-terminal loop and the alpha-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His(25), Lys(27), and Arg(41) of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala(20), Arg(21), Asn(30), and Lys(64). This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.     

Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.

BACKGROUND: Chemokines drive the migration of leukocytes via interaction with specific G protein-coupled 7-transmembrane receptors. The chemokine ligand/receptor pair stromal cell-derived factor-1 (SDF-1, CXCL12)/CXCR4 is gaining increasing interest because of its involvement in the metastasis of several types of cancer and in certain inflammatory autoimmune disorders such as rheumatoid arthritis. In addition, CXCR4 serves as an important coreceptor for cellular entry of T-tropic strains of human immunodeficiency virus (HIV). Therefore, potent and specific CXCR4 antagonists may have therapeutic potential as anti-HIV, anti-cancer, and anti-inflammatory drugs. METHODS AND RESULTS: Chemokine receptor antagonists can be identified by their ability to inhibit ligand binding to the receptor protein. Until now, chemokine binding assays were mostly performed with radiolabeled chemokine ligands such as [(125)I]CXCL12. To overcome the practical problems associated with such radioactive chemokine binding assays, we have developed a flow cytometric technique using a new, commercially available Alexa Fluor 647 conjugate of CXCL12 (CXCL12(AF647)). Calcium flux, chemotaxis, and p44/42 mitogen-activated protein kinase phosphorylation assays showed that the agonistic activity of the fluorescent CXCL12 was unchanged as compared with that of unlabeled CXCL12. Human T-lymphoid (CXCR4(+)) SupT1 cells and CXCR4-transfected, but not CCR5- or CXCR3-transfected, human astroglioma U87.CD4 cells specifically bound CXCL12(AF647) in a concentration-dependent manner. Unlabeled CXCL12 and the well-known CXCR4 inhibitors, AMD3100 and T22, blocked the binding of CXCL12(AF647) to SupT1 cells with 50% inhibitory concentrations of 92, 13, and 8 ng/ml, respectively. We have also used this method to evaluate CXCL12 binding and CXCR4 expression level in different subsets of human peripheral blood mononuclear cells. CONCLUSION: CXCL12(AF647) is a valuable, more convenient alternative for [(125)I]CXCL12 in ligand/receptor interaction studies.     

CXCL12-CXCL4 heterodimerization prevents CXCL12-driven breast cancer cell migration

Despite improvements in cancer early detection and treatment, metastatic breast cancer remains deadly. Current therapeutic approaches have very limited efficacy in patients with triple negative breast cancer. Among the many mechanisms associated that contribute to cancer progression, signaling through the CXCL12-CXCR4 is an essential step in cancer cell migration. We previously demonstrated the formation of CXCL12-CXCL4 heterodimers (Carlson et al., 2013). Here, we investigated whether CXCL12-CXCL4 heterodimers alter tumor cell migration. CXCL12 alone dose-dependently promoted the MDA-MB 231 cell migration (p < .05), which could be prevented by blocking the CXCR4 receptor. The addition of CXCL4 inhibited the CXCL12-induced cell migration (p < .05). Using NMR spectroscopy, we identified the CXCL4-CXCL12 binding interface. Moreover, we generated a CXCL4-derived peptide homolog of the binding interface that mimicked the activity of native CXCL4 protein. These results confirm the formation of CXCL12-CXCL4 heterodimers and their inhibitory effects on the migration of breast tumors cells. These findings suggest that specific peptides mimicking heterodimerization of CXCL12 might prevent breast cancer cell migration.     

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

CXCL12 signaling through G protein-coupled CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. The second CXCL12-receptor CXCR7 modulates the CXCL12/CXCR4 pathway by acting as a CXCL12 scavenger and exerts G protein-independent functions. Given the distinct properties of CXCR4 and CXCR7, we hypothesized that the distinct C-terminal domains differently regulate receptor trafficking and stability. Here, we examined epitope-tagged wild type and C-terminal mutant receptors in human embryonic kidney cells (HEK293) with respect to trafficking, stability, (125)I-CXCL12 degradation, and G protein-coupling. The 24 CXCR7 C-terminal residues were sufficient to promote rapid spontaneous internalization. Replacement of the CXCR7 C terminus with that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization and phosphorylation at the heterologous domain. The reverse tail-swap caused ligand-independent internalization of the resulting CXCR4-7tail mutant. Receptor-mediated (125)I-CXCL12 uptake and release of (125)I-CXCL12 degradation products were accelerated with receptors bearing the CXCR7 C terminus and impaired after conversion of CXCR7 C-terminal serine/threonine residues into alanines. C-terminal lysine residues were dispensable for plasma membrane targeting and the CXCL12 scavenger function but involved in constitutive degradation of CXCR7. Although the CXCR7 C terminus abolished G protein coupling in the CXCR4-7tail mutant, replacement of the CXCR7 C terminus, CXCR7 second intracellular loop, or both domains with the corresponding CXCR4 domain did not result in a G protein-coupled CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C terminus influences the ligand-uptake/degradation rate, G protein coupling, and receptor stability. Regulatory pathways targeting CXCR7 C-terminal serine/threonine sites may control the CXCL12 scavenger activity of CXCR7.     

Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12

Although chemokines are well established to function in immunity and endothelial cell activation and proliferation, a rapidly growing literature suggests that CXC Chemokine receptors CXCR3, CXCR4 and CXCR7 are critical in the development and progression of solid tumors. The effect of these chemokine receptors in tumorigenesis is mediated via interactions with shared ligands I-TAC (CXCL11) and SDF-1 (CXCL12). Over the last decade, CXCR4 has been extensively reported to be overexpressed in most human solid tumors and has earned considerable attention toward elucidating its role in cancer metastasis. To enrich the existing armamentarium of anti-cancerous agents, many inhibitors of CXCL12–CXCR4 axis have emerged as additional or alternative agents for neo-adjuvant treatments and even many of them are in preclinical and clinical stages of their development. However, the discovery of CXCR7 as another receptor for CXCL12 with rather high binding affinity and recent reports about its involvement in cancer progression, has questioned the potential of “selective blockade” of CXCR4 as cancer chemotherapeutics. Interestingly, CXCR7 can also bind another chemokine CXCL11, which is an established ligand for CXCR3. Recent reports have documented that CXCR3 and their ligands are overexpressed in different solid tumors and regulate tumor growth and metastasis. Therefore, it is important to consider the interactions and crosstalk between these three chemokine receptors and their ligand mediated signaling cascades for the development of effective anti-cancer therapies. Emerging evidence also indicates that these receptors are differentially expressed in tumor endothelial cells as well as in cancer stem cells, suggesting their direct role in regulating tumor angiogenesis and metastasis. In this review, we will focus on the signals mediated by this receptor trio via their shared ligands and their role in tumor growth and progression.     

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.     

Matrix metalloproteinase processing of CXCL11/I-TAC results in loss of chemoattractant activity and altered glycosaminoglycan binding

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.     

Imaging CXCL12-CXCR4 Signaling in Ovarian Cancer Therapy

Chemokine CXCL12 and receptor CXCR4 have emerged as promising therapeutic targets for ovarian cancer, a disease that continues to have a dismal prognosis. CXCL12-CXCR4 signaling drives proliferation, survival, and invasion of ovarian cancer cells, leading to tumor growth and metastasis. Pleiotropic effects of CXCR4 in multiple key steps in ovarian cancer suggest that blocking this pathway will improve outcomes for patients with this disease. To quantify CXCL12-CXCR4 signaling in cell-based assays and living mouse models of ovarian cancer, we developed a click beetle red luciferase complementation reporter that detects activation of CXCR4 based on recruitment of the cytosolic adapter protein β-arrestin 2. Both in two-dimensional and three-dimensional cell cultures, we established that bioluminescence from this reporter measures CXCL12-dependent activation of CXCR4 and inhibition of this pathway with AMD3100, a clinically-approved small molecule that blocks CXCL12-CXCR4 binding. We used this imaging system to quantify CXCL12-CXCR4 signaling in a mouse model of metastatic ovarian cancer and showed that treatment with AMD3100 interrupted this pathway in vivo. Combination therapy with AMD3100 and cisplatin significantly decreased tumor burden in mice, although differences in overall survival were not significantly greater than treatment with either agent as monotherapy. These studies establish a molecular imaging reporter system for analyzing CXCL12-CXCR4 signaling in ovarian cancer, which can be used to investigate biology and therapeutic targeting of this pathway in cell-based assays and living mice.     

Structural determinants involved in the regulation of CXCL14/BRAK expression by the 26 S proteasome

The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells; however, in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation.     

Quantitative phosphoproteomics of CXCL12 (SDF-1) signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.