Kank family proteins comprise a novel type of talin activator

We developed an integrative adhesome analysis by combining a focal adhesion enrichment index with integrin tail interactome to identify novel FA proteins. Using this approach we identified the evolutionarily conserved Kank protein family as novel FA proteins. Interestingly, Kank2 concentrates at the lateral border of FAs (which we term the FA belt), a previously unrecognized FA sub-compartment. Mechanistically, Kank directly binds the talin R7 domain through its evolutionarily conserved KN motif and promotes talin activation in a forceand F-actin-independent manner. Consequently, Kank2 induces and/or maintains the formation of active integrins and destabilizes talin-actin connection. Our data identify Kank family proteins as novel type of talin activator, which specifically stabilize integrin-talin complexes that are uncoupled from the actomyosin system. Introduction Mesenchymal migration relies on integrin receptors, a large family of α/β heterodimers that couple the extracellular matrix (ECM) to the actin cytoskeleton 1. To allow cell adhesion and migration, integrins have to be activated. Integrin activation is characterized by shifting the unbound form of the integrin from a low to a high affinity conformation followed by ligand binding 2. Ligand bound integrins then cluster and assemble a mechano-sensitive macro-protein complex (called the adhesome) around their cytoplasmic domains 3, 4. The initial adhesion complex are small, short-lived nascent adhesions (NAs) formed at the periphery of cell protrusions. A limited number of NAs subsequently couple to F-actin and actomyosin-mediated pulling forces reinforce the integrin-ligand interaction and promote the recruitment of more proteins to form large, mature focal adhesions (FAs). The dynamic engagement between integrin and actomyosin serves as a molecular clutch that promotes both forward membrane protrusion and rearward traction force to drive cell migration 5. Talin and kindlin directly bind to β integrin cytoplasmic tails and play key roles in integrin activation and coupling to actin 6. Talin is a large, ~270 kD protein composed of an Nterminal FERM (protein 4.1, ezrin, radixin, moesin) domain, also called talin head domain (THD), and a long C-terminal rod domain consisting of 13 helical bundles (R1-R13 domains). The THD is divided into four subdomains (F0, F1, F2, F3). The F3 subdomain of the THD binds β integrin tails and negatively charged lipids on the plasma membrane, and mediates integrin activation. The rod domain contains two F-actin binding sites, several binding sites for RIAM, numerous binding sites for vinculin and a dimerization domain at the extreme C-terminus 7. The F-actin binding through talin rod couples the integrin/talin complexes to actomyosin, which is required for adhesion re-enforcement and rigidity sensing. Talin cycles between plasma membrane, where it activates integrins, and cytosol, where it adopts an auto-inhibited form, presumably as a compact globular dimer 8 with an intramolecular interaction between the F3 subdomain and the R9 domain 9, 10. Since this interaction masks the integrin binding site, talin requires an activation step that disrupts the auto-inhibitory head-rod interaction. A prime candidate for talin activation is the Rap1 effector protein RIAM (Rap1–GTP-interacting adapter molecule), which binds the R8 helical bundle 11, relieves the autoinhibitory interaction and recruits talin to the plasma membrane 12, 13. A second, potential talin activator is PtdIns(4,5)P2 10. A splice isoform of phosphatidylinositol 4-phosphate 5-kinase type Iγ (PIPKIγ90) locally synthesizes PtdIns(4,5)P2 upon recruitment to FAs through direct binding to talin F3 subdomain 14. Finally, the actin retrograde flow was shown to induce the high affinity state of αLβ2 (LFA1) on T cells 15, although it is not clear whether talin is the target of the actin flow. However, it is clear from genetic studies that talin activation is a highly redundant process, as neither RIAMnor PIPKIγ90-deficient mice have apparent phenotypes 16, 17. In the present paper we developed a FA enrichment index to identify novel FA proteins through high-resolution quantitative mass spectrometry (MS). We identified the evolutionarily conserved Kank protein family as novel FA proteins. Kank family proteins consist of 4 members (Kank1-4) characterized by an N-terminal Kank (KN) motif, several central coil-coil domains and C-terminal ankyrin repeats 18. Kank1 was shown to restrict microtubule outgrowth at cell cortex by recruiting Kif21a to liprin β1 19. Overexpressed Kank proteins inhibit stress fiber formation and migration probably by downregulating RhoA 20. A single Kank ortholog in C.elegans, called VAB19, controls epidermis-muscle attachment 21, neuronal migration 22 and basement membrane sliding during vulval morphogenesis 23. The Kank3 knockdown in zebrafish leads to epidermal detachment 24. We found that Kank localizes to the outer border of FAs (which we term the FA belt) and to central fibrillar adhesion, and induces and/or maintains the formation of active integrins at sites lacking actomyosin coupling. Mechanistically, Kank directly binds the talin R7 domain through its evolutionarily conserved KN motif and promotes talin activation in a forceand F-actin-independent manner. Our data identify Kank as novel talin activator, which specifically stabilizes integrin-talin complex that are uncoupled from the actomyosin