Structural studies of IPK1: how molecular turtles are made

Inositol phosphates (IPs) are signaling molecules implicated in a variety of cellular processes, notably, cell survival signaling and vesicular trafficking, which underlie diseases such as cancer and diabetes. The specific roles of many IPs in disease states have yet to be determined. Inositol phosphate kinases (IPKs) phosphorylate inositol 1,4,5-trisphosphate (IP3) on different positions of its inositol ring to yield an array of unique IPs. Control of IP production at different stages of the IP metabolic pathway could be used as an approach to determine the functional roles of each IP. In this thesis, we focus on inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), which phosphorylates IP5 to IP6. These two IPs regulate apoptosis in vitro and in vivo, revealing a role for IPK1 in cell death, but their precise mechanisms of action remain unresolved. Our overall goal was to structurally and biochemically characterize IPK1 to identify how it selects its substrates, how it is regulated, and how it may be targeted with small molecules to be used as tools to study IPK1 function. We determined the IP-free crystal structure of IPK1, which revealed that the N-lobe of IPK1 is unstable in the absence of substrate. Based on this observation, we hypothesized that IPK1 uses a mechanism of IP-induced stabilization to select IP5 as its substrate: IP5 is initially recognized by IPK1 through the 4-, 5-, and 6-phosphates, and then, the 1- and 3-phosphates induce N-lobe stabilization, thereby allowing IPK1 activation only when the appropriate IP is bound. The key interaction between R130 and the 1-phosphate of the IP stabilizes the N-lobe for subsequent kinase activation. To validate our hypothesis, we evaluated the role of each IP phosphate for IP binding and kinase activation. We determined that the 5- and 6-phosphates were more important for IP binding, while the 1- and 3-phosphates were more important for IPK1 activation. Moreover, we demonstrated that IPs lacking the 1- or 3-phosphates were unable to stabilize IPK1 to the same extent as IP5, and that artificial stabilization of the N-lobe by engineered disulfide bonds altered IPK1 substrate specificity, by reducing the need for N-lobe interactions with substrate. We also characterized PKRnc and Catechin Gallate as leads for the development of small molecule inhibitors of IPK1. Taken together, our studies provide a basis for the development of selective inhibitors for IPK1 to investigate the roles of IPs that modulate apoptotic signaling pathways. Moreover, IP-induced stabilization distinguishes IPK1 from other IPKs and provides important considerations for the selective inhibition of each IPK. Uncovering the role of IPs in different cellular processes may ultimately lead to novel treatments for diseases whose underlying mechanisms are mediated by IP signaling.

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