Figure 2: ANKS6 exerts its kinase stimulating activity through direct interaction with the NEK8 kinase domain.
From: ANKS6 is the critical activator of NEK8 kinase in embryonic situs determination and organ patterning
(a) NEK8 truncations were employed in IP/binding assays, to map interaction sites. (b) The kinase domain of NEK8, along with a short C-terminal extension (NEK81-295) mediates the interaction with ANKS6, while the RCC1 domain does not contribute to complex formation. (c) NEK8 interacts with inversin through its RCC1 domain; the five C-terminal RCC1 repeats (amino acids 416–698), in particular, are necessary and sufficient for binding. (d) The NEK8 kinase domain (NEK81-258), as predicted by homology alignment, is not capable of binding ANKS6 and is inactive as a kinase. NEK81-295, a larger truncation that binds ANKS6, exhibits dramatically enhanced phosphorylation activity. The full-length wild-type NEK8 and the NEK81-415 truncation are included as controls. Any phosphorylation is only detected in the presence of ANKS6, generating autoradiographic signals at the levels of ANKS6 (100 kDa), α-casein (25 kDa) and the molecular weight of the respective NEK8 truncations. (e) Co-IP of myc-ANKS6 with FLAG-NEK8, -INVS and -NPHP3, and fractionation with different ionic strength conditions demonstrates formation of a salt-insensitive robust subcomplex between NEK8 and ANKS6.
