TY  - GEN
T1  - Missense mutation of a conserved residue in UNC-112 (kindlin) eliminates binding to PAT-4 (ILK)
AU  - Qadota, Hiroshi
AU  - Oberhauser, Andres F
AU  - Benian, Guy M
DO  - 10.17912/micropub.biology.000454
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000454/
AB  - C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly (Rogalski et al. 2000; Meves et al. 2009), and is one component of a conserved four-protein complex that associates with the cytoplasmic tail of integrin at the base of integrin adhesion complexes in muscle (Mackinnon et al. 2002; Lin et al. 2003; Norman et al. 2007; Qadota et al. 2014). UNC-112 binds directly with the cytoplasmic tail of PAT-3 (beta-integrin)(Qadota et al. 2012). UNC-112 binds to PAT-4 (ILK)(Mackinnon et al. 2002), and PAT-4 binds to both PAT-6 (alpha-parvin)(Lin et al. 2003), and UNC-97 (PINCH)(Mackinnon et al. 2002; Norman et al. 2007). A complex consisting of UNC-112, PAT-4, PAT-6 and UNC-97 has been demonstrated by co-immunoprecipitation (Qadota et al. 2014). UNC-112 is comprised of 720 amino acid residues and contains FERM and PH domains. The N-terminal half of UNC-112 (1-396 aa) can bind to the C-terminal half of UNC-112 (397-720 aa), and this interaction is inhibited by the association of PAT-4 (ILK) to the N-terminal half of UNC-112 (Qadota et al. 2012). In support of this model, we identified a D382V mutation that results in lack of binding to PAT-4 (Qadota et al. 2012). However, this residue is not conserved in human Kindlins (Figure 1D; D in UNC-112 but S in Kindlins), and mutation of this S to V in Kindlin-2 did not inhibit ILK binding (Huet-Calderwood et al. 2014). Here, we report identification of a novel UNC-112 mutation of a conserved residue that cannot bind to PAT-4. We found that E302G UNC-112 cannot bind to PAT-4, but still can bind to PAT-3 (beta-integrin) (Figure 1A). When we expressed HA tagged UNC-112 with E302G in C. elegans muscle (Figure 1B), HA tagged E302G UNC-112 cannot localize to the integrin adhesion complexes (dense bodies and M-lines) (Figure 1C). The E302 residue of UNC-112 is conserved in human Kindlin-1 and Kindlin-2, but not in Kindlin-3 (Figure 1D). Based on the only available crystal structure for a kindlin, that being for human kindlin-3 (Sun et al. 2020; Bu et al. 2020), we generated a homology model of UNC-112 and highlighted the locations of E302 and D382 (Figure 1E). Both E302 and D382 are located on surface loops of the structure. Mutating E302 to G, or mutating D382 to V resulted in no clashes with neighboring residues, based on the rotamer mutagenesis and energy minimization tools in Chimera (Pettersen et al., 2004). In fact, the lack of a sidechain of G, or the smaller sidechain of V, is likely to make these loops even more flexible. These mutations are predicted to not alter the overall structure of UNC-112 but could possibly affect the surface binding of UNC-112 to PAT-4. It should be noted, that in addition to our findings, a L located 6 residues C-terminal of D382, within the same FERM_M domain, and conserved in UNC-112 and all human kindlins (Figure 1D), when mutated to A, also greatly reduces binding to ILK (Huet-Calderwood et al. 2014). However, the results by Huet-Calderwood et al. were obtained by GST pulldown from lysates of tissue culture cells that overexpress both ILK and alpha-parvin. Our results, using the yeast two hybrid method and localization in worm muscle cells in which only UNC-112 was overexpressed, provide more evidence for an effect on direct binding between UNC-112 (kindlin) and PAT-4 (ILK). Our finding of a conserved residue in UNC-112 in the FERM_N domain that reduces binding to PAT-4, opens the door to testing the comparable residues in human Kindlin-1 and Kindlin-2 for their importance to binding to ILK. We do not know what the phenotype is of a nematode that is homozygous for the UNC-112 E302G mutation. Since UNC-112 function requires PAT-4 (Mackinnon et al. 2002), and all known mutations in pat-4 are Pat embryonic lethal, worms homozygous for UNC-112 E302G, might be Pat embryonic lethal. Several mutations in unc-112 are known to result in either the Pat embryonic phenotype or the adult viable Unc phenotype in which adults move slowly and have a disorganized myofilament lattice (Rogalski et al. 2000). The three known Pat alleles are unc-112(st562) and unc-112(st581) that are both nonsense mutations, and unc-112(gk1) that is a deletion. The Unc allele, unc-112(r367), is a missense mutation, T85I, and is also temperature sensitive. The molecular properties of UNC-112 T85I have not been explored, and thus, we do not know if the phenotype is due to reduced binding to PAT-4 or PAT-3, or whether it results in a unstable protein. Finally, there is one unusual allele created by CRISPR/Cas9, unc-112(kq715), L715E, which shows a defect in the migration of the distal tip cell (Park et al. 2020), but whether there was an effect on the myofilament lattice of body wall muscle, was not reported.
PY  - 2021
JO  - microPublication Biology
ER  -