Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain
Tóm tắt
Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4. We immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127–602) or the extracellular domain plus the transmembrane domain (Aa 106–602). Positive binding was detected to both CKAP4126–602 and CKAP4106–602, suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4127–360 and CKAP4361–524 exhibited a fast association rate (k
on
) and a slow dissociation rate (k
off
), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127–524 region of the CKAP4 extracellular domain. We determined that the CKAP4127–360 and CKAP4361–524 mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.
Tài liệu tham khảo
Keay S, Kleinberg M, Zhang CO, Hise MK, Warren JW. Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production. J Urol. 2000;164(6):2112–8.
Keay SK, Szekely Z, Conrads TP, Veenstra TD, Barchi JJ Jr, Zhang CO, Koch KR, Michejda CJ. An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci U S A. 2004;101(32):11803–8.
Conrads TP, Tocci GM, Hood BL, Zhang CO, Guo L, Koch KR, Michejda CJ, Veenstra TD, Keay SK. Ckap4/p63 Is a receptor for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis patients. J Biol Chem. 2006;281(49):37836–43.
Schweizer A, Ericsson M, Bachi T, Griffiths G, Hauri HP. Characterization of a novel 63 kda membrane protein. Implications for the organization of the er-to-golgi pathway. J Cell Sci. 1993;104(Pt 3):671–83.
Schweizer A, Rohrer J, Hauri HP, Kornfeld S. Retention of p63 in an er-golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers. J Cell Biol. 1994;126(1):25–39.
Klopfenstein DR, Kappeler F, Hauri HP. A novel direct interaction of endoplasmic reticulum with microtubules. EMBO J. 1998;17(21):6168–77.
Vedrenne C, Hauri HP. Morphogenesis of the endoplasmic reticulum: beyond active membrane expansion. Traffic. 2006;7(6):639–46.
Vedrenne C, Klopfenstein DR, Hauri HP. Phosphorylation controls climp-63-mediated anchoring of the endoplasmic reticulum to microtubules. Mol Biol Cell. 2005;16(4):1928–37.
Planey SL, Zacharias DA. Palmitoyl acyltransferases, their substrates, and novel assays to connect them (review). Mol Membr Biol. 2009;26(1):14–31.
Shahjee HM, Koch KR, Guo L, Zhang CO, Keay SK. Antiproliferative factor decreases akt phosphorylation and alters gene expression via ckap4 in t24 bladder carcinoma cells. J Exp Clin Cancer Res. 2010;29:160.
Matika CA, Wasilewski M, Arnott JA, Planey SL. Antiproliferative factor regulates connective tissue growth factor (ctgf/ccn2) expression in t24 bladder carcinoma cells. Mol Biol Cell. 2012;23(10):1976–85.
Razzaq TM, Bass R, Vines DJ, Werner F, Whawell SA, Ellis V. Functional regulation of tissue plasminogen activator on the surface of vascular smooth muscle cells by the type-ii transmembrane protein p63 (ckap4). J Biol Chem. 2003;278(43):42679–85.
Gupta N, Manevich Y, Kazi AS, Tao JQ, Fisher AB, Bates SR. Identification and characterization of p63 (ckap4/ergic-63/climp-63), a surfactant protein a binding protein, on type ii pneumocytes. Am J Physiol Lung Cell Mol Physiol. 2006;291(3):L436–46.
Bates SR, Kazi AS, Tao JQ, Yu KJ, Gonder DS, Feinstein SI, Fisher AB. Role of p63 (ckap4) in binding of surfactant protein-a to type ii pneumocytes. Am J Physiol Lung Cell Mol Physiol. 2008;295(4):L658–69.
Kimura H, Fumoto K, Shojima K, Nojima S, Osugi Y, Tomihara H, Eguchi H, Shintani Y, Endo H, Inoue M, Doki Y, et al. Ckap4 Is a dickkopf1 receptor and is involved in tumor progression. J Clin Invest. 2016;126(7):2689–705.
Kikuchi A, Fumoto K, Kimura H. The dickkopf1-ckap4 axis creates a novel signaling pathway and may represent a molecular target for cancer therapy. Br J Pharmacol. 2017;
Planey SL, Keay SK, Zhang CO, Zacharias DA. Palmitoylation of cytoskeleton associated protein 4 by dhhc2 regulates antiproliferative factor-mediated signaling. Mol Biol Cell. 2009;20(5):1454–63.
Zhang J, Planey SL, Ceballos C, Stevens SM Jr, Keay SK, Zacharias DA. Identification of ckap4/p63 as a major substrate of the palmitoyl acyltransferase dhhc2, a putative tumor suppressor, using a novel proteomics method. Mol Cell Proteomics. 2008;7(7):1378–88.
Ling J, Liao H, Clark R, Wong MS, Lo DD. Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance. J Biol Chem. 2008;283(45):30585–95.
Haas J, Roth S, Arnold K, Kiefer F, Schmidt T, Bordoli L, Schwede T: The protein model portal--a comprehensive resource for protein structure and model information. Database (Oxford) 2013;2013:bat031.
Roy A, Kucukural A, Zhang Y. I-tasser: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010;5(4):725–38.
Barchi JJ Jr, Kaczmarek P. Short and sweet: evolution of a small glycopeptide from a bladder disorder to an anticancer lead. Mol Interv. 2009;9(1):14–7.
Kaczmarek P, Tocci GM, Keay SK, Adams KM, Zhang CO, Koch KR, Grkovic D, Guo L, Michejda CJ, Barchi JJ Jr. Structure-activity studies on antiproliferative factor (apf) glycooctapeptide derivatives. ACS Med Chem Lett. 2010;1(8):390–4.
Mallajosyula SS, Adams KM, Barchi JJ, MacKerell AD. Conformational determinants of the activity of antiproliferative factor glycopeptide. J Chem Inf Model. 2013;53(5):1127–37.
Kaczmarek P, Keay SK, Tocci GM, Koch KR, Zhang CO, Barchi JJ Jr, Grkovic D, Guo L, Michejda CJ. Structure-activity relationship studies for the peptide portion of the bladder epithelial cell antiproliferative factor from interstitial cystitis patients. J Med Chem. 2008;51(19):5974–83.
Keay S, Zhang CO, Hise MK, Hebel JR, Jacobs SC, Gordon D, Whitmore K, Bodison S, Gordon N, Warren JW. A diagnostic in vitro urine assay for interstitial cystitis. Urology. 1998;52(6):974–8.
Keay S, Zhang CO, Marvel R, Chai T. Antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor: sensitive and specific urine markers for interstitial cystitis. Urology. 2001;57(6 Suppl 1):104.
Keay S, Zhang CO, Shoenfelt JL, Chai TC. Decreased in vitro proliferation of bladder epithelial cells from patients with interstitial cystitis. Urology. 2003;61(6):1278–84.