WFIKKN1 and WFIKKN2 bind growth factors TGFb1, BMP2 and BMP4 but do not inhibit their signalling activity Gyo¨ rgy Szla´ ma, Katalin Konda´ s, Ma´ ria Trexler and La´ szlo´ Patthy Institute of Enzymology, Budapest, Hungary Keywords WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins BMP; GDF11; GDF8; TGFb; WFIKKN consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Correspondence Recent experiments have shown that both proteins have high affinity for L. Patthy, Institute of Enzymology, Budapest, Karolina ut 29, Hungary growth and differentiation factor (GDF)8 and GDF11. Here we study the Fax: +361 466 5465 interaction of WFIKKN proteins with several additional representatives of Tel: + 361 209 3537 the transforming growth factor (TGF)b family using SPR measurements. E-mail: [email protected] Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFb1, bone morphogenetic pro- (Received 1 June 2010, revised 5 October )6 tein (BMP)2 and BMP4 with relatively high affinity (Kd 10 M). To 2010, accepted 8 October 2010) assess the biological significance of these interactions we studied the effect doi:10.1111/j.1742-4658.2010.07909.x of WFIKKN proteins on the activity of GDF8, GDF11, TGFb1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFb1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFb1, BMP2 and BMP4 they function as growth factor binding pro- teins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to estab- lish growth factor gradients in the extracellular space. Structured digital abstract l A list of the large number of protein-protein interactions described in this article is available via the MINT article MINT-8044119 family are usually assigned to three main subfamilies: Introduction activins, TGFbs and bone morphogenic proteins Growth factors of the transforming growth factor b (BMPs) ⁄ growth and differentiation factors (GDFs). (TGFb) family regulate many cellular processes, includ- TGFb family members are secreted as large precursor ing cell proliferation, differentiation and lineage deter- proteins and the mature growth factors are released from mination. In humans, more than 30 structurally related these precursors through cleavage by furin-type prote- proteins belong to this family [1]. Members of this protein ases. In several cases, the prodomain and the mature Abbreviations ACRIIB, activin receptor IIB; BMP2, bone morphogenetic protein 2; BMP3, bone morphogenetic protein 3; BMP4, bone morphogenetic protein 4; BMP8b, bone morphogenetic protein 8b; BMP11, bone morphogenetic protein 11 or growth and differentiation factor 11; BMPRIA, bone morphogenetic protein receptor IA; ECD, extracellular domain; GDF11, growth and differentiation factor 11 or bone morphogenetic protein 11; GDF8, growth and differentiation factor 8 or myostatin; TGFb1, transforming growth factor b1; TGF-bsRII, recombinant protein corresponding to the extracellular domain of TGFb1 receptor TGF-bbRII; WFIKKN1 and WFIKKN2 – WAP, follistatin, immunoglobulin, kunitz and netrin domain containing protein 1 and 2. 5040 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS G. Szla´ma et al. WFIKKNs bind several members of the TGFb family disulfide-bonded homodimer growth factor remain asso- in the micromolar range. Our data suggest that ciated after proteolytic cleavage [2–4]. The prodo- WFIKKN proteins may function not only as antago- main ⁄ growth factor complexes confer latency on the nists of GDF8 and GDF11, but also as proteins that growth factors and the active homodimeric growth fac- localize the action of growth factors. tors may be liberated from the latent complexes through degradation of the propeptides by proteases [4–6]. Results TGFb family proteins signal through type I and type II serine–threonine kinase receptors; in verte- Characterization of the interaction of WFIKKN1 brates, seven type I receptors and five type II receptors and WFIKKN2 with BMP2, BMP3, BMP4, BMP8b have been identified [1]. Homodimeric growth factors and TGFb1 by SPR bind to two type I and two type II receptors to form a hexameric signalling complex. In these complexes, SPR analyses suggested that both WFIKKN proteins type II receptors phosphorylate a short segment of may bind BMP2, BMP3, BMP4, BMP8b and TGFb1 type I receptors, which in turn phosphorylate down- (Fig. 1), although the affinities of WFIKKN1 and stream targets [7,8]. WFIKKN2 for these growth factors are significantly The number of growth factors available for signal- lower than those determined for GDF8 and GDF11 ling is tightly regulated by several, structurally differ- (Table 1). No interaction was detected with activin A, ent antagonists that, by interacting with the growth even when high (up to 4 lm) concentrations of factors, alter or diminish their binding to the receptors. WFIKKNs were injected on the surface of immobi- Similar to the prodomain in latent complexes, inhibi- lized activin A. tory proteins, like chordin, noggin, follistatin, follista- The Kd values calculated for the interactions of ) tin-related protein and gremlin bind various members WFIKKN1 with BMP2 (7.2 · 10 7 m), BMP3 (3.3 · ) ) ) of the TGFb family with high affinity and block their 10 6 m), BMP4 (8.2 · 10 7 m), TGFb1 (4.5 · 10 7 m) interaction with their receptors [9]. or for the interactions of WFIKKN2 with BMP2 ) ) ) Recent studies have expanded the list of TGFb (4.3 · 10 8 m), BMP3 (1.8 · 10 7 m), BMP4 (6.5 · 10 8 ) antagonists to include WFIKKN1 and WFIKKN2 m), TGFb1 (2.8 · 10 8 m) were suggestive of relatively proteins: these proteins bind GDF8 (myostatin) and high affinities, raising the possibility that these interac- GDF11 (BMP11) with high affinity [10]. WFIKKN tions may have biological importance. proteins are large extracellular multidomain proteins It should be noted, however, that there was a major that contain a WAP domain, a Follistatin ⁄ Kazal additional difference between sensorgrams obtained domain, an immunoglobulin domain, two Kunitz-type with GDF8 ⁄ GDF11 and TGFb1 ⁄ BMP2 ⁄ BMP3⁄ BMP4⁄ protease inhibitor domains and an NTR domain BMP8b: the former gave good fits with the simple [11,12]. The fact that, in luciferase reporter assays, model of a 1 : 1 Langmuir interaction [10], whereas the WFIKKN2 inhibited the activity of myostatin and association and dissociation curves of the interaction of GDF11 [13] suggests that WFIKKNs may play crucial WFIKKN proteins with the various BMPs gave accept- roles in the regulation of processes (muscle growth, able fits only with the model of ‘two state reaction with anterior ⁄ posterior patterning of the axial skeleton, etc.) conformational change’. The association and dissocia- that are under the control of these growth factors. tion curves of the interaction of WFIKKN proteins WFIKKN proteins are, however, expressed in with TGFb1 could be fitted to the model of ‘heteroge- numerous tissues other than those controlled by GDF8 neous ligand parallel reaction’ (see Experimental proce- or GDF11. For example, the WFIKKN1 gene is dures). Because it has been pointed out recently in a expressed in pancreas, thymus, liver, kidney, lung, tes- critical review of the biosensor literature that parame- tis and inner ear, and the WFIKKN2 gene is expressed ters calculated with the 1 : 1 interaction model are most in ovary, testis, pancreas, brain and lung [11,12,14], likely to give reliable estimates of binding constants raising the possibility that the proteins may have addi- [15], it may be doubtful whether the Kd values calcu- tional functions. To investigate this possibility, we used lated for the interaction of WFIKKN proteins with SPR and luciferase reporter assays to study the inter- TGFb1, BMP2, BMP3, BMP4, BMP8b are valid, and action of WFIKKN1 and WFIKKN2 proteins with whether WFIKKNs are efficient inhibitors of the bind- several representatives of the TGFb family. ing of these growth factors to their cognate receptors. Analyses of SPR sensorgrams have shown that both To answer these questions, we studied the ability of WFIKKN proteins bind TGFb1, BMP2 and BMP4 WFIKKN proteins to block the binding of growth fac- )6 with relatively high affinity (Kd 10 m), but in tors to their receptors using SPR in a solution-competi- reporter assays they do not inhibit their activities, even tion format, as well as luciferase reporter assays. FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS 5041 WFIKKNs bind several members of the TGFb family G. Szla´ma et al. WFIKKN1-BMP4 WFIKKN2-BMP4 A B 380 100 220 RU 20 60 0 200 400 600 800 0 200 400 600 800 Time (s) Time (s) WFIKKN1-TGFβ1WFIKKN2-TGF1 C 100 D 220 60 160 RU 100 20 40 0 200 400 600 800 0 200 400 600 800 Time (s) Time (s) E WFIKKN1-BMP2F WFIKKN2-BMP2 700 380 460 220 RU 220 60 0 200 400 600 800 0 200 400 600 800 Time (s) Time (s) G WFIKKN1-BMP3H WFIKKN2-BMP3 300 950 140 RU550 RU RU RU RU 150 0 200 400 600 800 0 200 400 600 800 Time (s) Time (s ) WFIKKN1-BMP8b WFIKKN2-BMP8b I J 620 80 RU 300 40 RU 0 0 200 400 600 800 0 200 400 600 800 Time (s) Time (s) Fig. 1. Characterization of the interaction of WFIKKN1 and WFIKKN2 with members of the TGFb family using SPR assays. Sensorgrams of the interactions of: (A) WFIKKN1 (500 nM, 1, 1.25, 1.5 and 2 lM) with BMP4; (B) WFIKKN2 (25, 50, 100, 250, 500 and 1000 nM) with BMP4; (C) WFIKKN1 (1, 2, 3, 4 and 6 lM) with TGFb1; (D) WFIKKN2 (250 AND 500 nM, and 1, 2 and 4 lM) with TGFb1; (E) WFIKKN1 (400 and 750 nM, 2 and 3 lM) with BMP2; (F) WFIKKN2 (50, 100, 250 and 500 nM) with BMP2; (G) WFIKKN1 (500 and 750 nM, 1 and 1.5 lM) with BMP3; (H) WFIKKN2 (100, 250 and 500 nM, 1 and 2 lM) with BMP3; (I) WFIKKN1 (250, 500 and 750 nM, 1, 1.25, 1.5 and 2 lM) with BMP8b; (J) WFIKKN2 (250, 500 and 750 nM, 1 and 1.5 lM) with BMP8b.