bioRxiv preprint doi: https://doi.org/10.1101/524447; this version posted January 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Integrin cytoplasmic domain and pITAM compete for spleen tyrosine kinase binding Lina Antenucci1, Maarit Hellman2, Vesa P. Hytönen3, Perttu Permi1,2, and Jari Ylänne1* From the 1Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Survontie 9 C, P.O. BOX 35, FI-40014 Jyväskylä, Finland; 2Department of Chemistry and Nanoscience Center, University of Jyväskylä, Survontie 9 C, P.O. BOX 35, FI-40014 Jyväskylä, Finland; 3Faculty of Medicine and Health Technology and BioMediTech, Tampere University, and Fimlab Laboratories, FI-33014 Tampere, Finland Running title: Competition of integrin and pITAM for Syk binding Author ORCID ID codes: LA: 0000-0003-1201-9342; VPH 0000-0002-9357-1480; PP 0000-0002-6281-1138; JY: 0000- 0003-4627-021X *To whom correspondence should be addressed: Jari Ylänne Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Survontie 9 C, P.O. BOX 35, FI-40014 Jyväskylä, Finland; [email protected]; Tel +350-504285273 Keywords: Syk, pITAM, SH2 domain, phosphotyrosine, NMR ABSTRACT understanding the function of cellular signaling networks. All non-receptor tyrosine kinases share In hematopoietic tissues cell-cell a similar enzymatically active kinase domain but communication involves immunoreceptors and their regulatory domains allow their classification specialized cell adhesion receptors that both to 10 functional groups (2). Spleen tyrosine kinase mediate intracellular signals. Spleen tyrosine (Syk) is a non-receptor tyrosine kinase that belongs kinase (Syk) is a non-receptor tyrosine kinase to a distinct kinase subfamily whose regulatory involved in the downstream signaling of both domain is composed of two phosphotyrosine- immunoreceptors tyrosine activation motif binding Src homology 2 (SH2) domains (3).The (ITAM) receptors and integrin family cell adhesion other member of this subfamily in mammals is the receptors. Both phosphorylated ITAM (pITAM) zeta-chain associated kinase of 70 kD (ZAP70) (3). and integrins bind to the regulatory domain of Syk Gene depletion studies in mouse have shown that composed of two Src homology 2 (SH2) domains. Syk is specifically required for the development of The interaction with pITAM is mediated by B-cells (4, 5) and ZAP70 for T-cell maturation (6, binding of a specific phosphotyrosine to each of the 7). Syk-deficient mice die newborn because of SH2 domains, leading to conformational changes failure to separate blood vessels and lymphatic and Syk kinase activation. Integrins bind to the vessels (4, 5), a defect that may reflect the role of interdomain A segment between the two SH2 platelet adhesion in this process (3). In addition to domains and to the N-terminal SH2 domain, but the platelets and lymphocytes, Syk has essential detailed binding site is not known. In order to map function in many other cells of hematopoietic the binding site, we performed NMR titration origin as well as in many non-hematopoietic experiments. We found that integrin cytoplasmic tissues, for instance in breast cancers (8). This domain peptide induced chemical shift changes reflects its function in mediating signal near the IA segment and at the phosphotyrosine transduction from adaptive immune receptors, binding site of the N-terminal SH2 domain of Syk. innate immune receptors and certain cell adhesion These changes were distinct, but partially receptors. overlapping with those induced by pITAM peptide. The mechanism of Syk activation We were also able to show that pITAM peptide downstream of most of the above listed receptors inhibited integrin binding to Syk regulatory is rather well understood: Src family kinases (Fyn domain. These results suggest that ITAM receptors and Lck) are activated upon immune receptor and integrins cannot bind simultaneously to Syk, clustering and phosphorylate two Tyr residues in but provide two distinct routes for Syk activation. the cytoplasmic domains of the immune receptor _____________________________________ complex (reviewed in (3)). These residues are found in a conserved sequence motif called the Tyrosine kinases are an important class of immunoreceptor tyrosine-containing activation cellular signaling enzymes and main targets of motif (ITAM) that has a consensus sequence: several cancer drugs (1). Understanding the YxxL/Ix6-12YxxL/I. ITAM is found either in the regulation of tyrosine kinases is a key for main transmembrane spanning polypeptide of the 1 bioRxiv preprint doi: https://doi.org/10.1101/524447; this version posted January 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. immune receptor (as in FcγRIIA) or in a separate pITAM interaction with SH2 domains mostly transmembrane adaptor in immune receptors (as in follow the general mode of pTyr/SH2 interaction TCR, BCR and FcεRI), innate immune pattern- (9, 19). The main interaction is mediated by the recognizing receptors (in certain C-type lectin phosphate group of the pTyr residue and in receptors as CLEC4E, CLEC6A, CLEC5A), or cell addition hydrophobic interaction with the +3 adhesion receptors (in platelet glycoprotein IV, position Ile or Leu residue determines the GPIV, and osteoclasts-associated receptor, specificity of the interaction (19). The exact OSCAR). In addition, C-type lectin receptors residues of integrin tail mediating the contact with exists where the full ITAM motif is formed by Syk regulatory domain are not known, but clustering of receptors containing single truncation studies have shown that the deletion of phosphorylated tyrosine motifs, so called four C-terminal residues in β3 abolish the hemITAM motifs (CLEC2, CLEC7A, CLEC9A) interaction (12) and that the minimum β3 (reviewed in (3)). Phosphorylated ITAM (pITAM) cytoplasmic tail peptide is Arg734-Thr762 (29 sequences are able to interact with the two SH2 residues) (12). On the Syk side, the main domains of Syk so that each of the pTyr residues interaction sites have been mapped to the binds to one SH2 domain (9). This interaction interdomain A (IA) segment and the N-terminal changes the relative orientation of these two SH2 domain (13) domains resulting in the detachment of We have earlier shown that binding of interdomain A (IA) and interdomain B (IB) soluble β3 integrin cytoplasmic domain does not segment from the kinase domain leading to kinase directly activate Syk as in the case of soluble domain activation (9, 10). pITAM, but clustered integrin peptides can In contract to phosphorylation-dependent activate purified Syk (20). This fits well with the interaction with transmembrane receptors or idea that integrins do not bind to the two SH2 adaptors, described above, Syk interacts with domains in a similar way as pITAM and thus they integrin family cell adhesion receptors cannot induce similar reorientation of Syk SH2 independently on receptor phosphorylation (11, domains as pITAM. 12). Direct interaction has been shown at least with In this study we set to test the hypothesis β1, β2 and β3 integrin subunit cytoplasmic domains that integrin and pITAM binding to Syk are (12, 13). Integrin cytoplasmic domains contain Tyr independent on each other. We used surface residues, but they do not form ITAM motifs and plasmon resonance (SPR) and nuclear magnetic Tyr phosphorylation of integrin tails inhibits Syk resonance (NMR) spectroscopy methods to interaction (12, 13). compare the binding surfaces of pITAM and β3 In many cell adhesion events ITAM integrin cytoplasmic domain on Syk regulatory signaling and integrin signaling happens domains. We find that the Syk IA segment is the simultaneously and sometimes even in the same main binding site of integrin, but on the N-terminal adhesion complex. For instance, during platelet SH2 domain, binding of integrin and pITAM adhesion to vessel wall, ITAM-dependent peptides induce NMR chemical shift perturbations signaling from the GPIV collagen receptor may (CSPs) on overlapping areas. Interestingly, the happen simultaneously with the function of the integrin responsive surface on N-SH2 is close to integrin α2β1 collagen receptor (14) and is the location of the IA in published structures. We immediately followed by the function of integrin also show that soluble pITAM inhibits Syk αIIbβ3, which is the major fibrinogen receptor regulatory domain binding to integrin-coated mediating platelet aggregation but also interact surfaces. We believe these findings are important with several other extracellular matrix proteins for understanding the cross-talk between integrins (15). In immunological synapses TCR and αLβ2 and ITAM receptors in Syk signaling. integrins function simultaneously in specific adhesion zones and signaling from these zones or Results cluster is regulated by diverse kinases and phosphatases (16). During osteoclast adhesion to Mapping of pITAM and integrin binding bones, ITAM-linked OSCAR and integrin αVβ3 surfaces on the N-terminal SH2 domain of Syk signal parallelly (17). To be able to study the possible cross-talk While the pITAM-Syk interaction between