Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation

Abstract

Background: The mechanism of substrate recognition for IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) is unresolved.Results: Binding and activity data reveal specific roles for each phosphate of IP5.Conclusion: The phosphate profile of IP5 is mechanistically critical to IPK1 activation.Significance: Identifying determinants of substrate specificity will aid in the design of selective inhibitors for IPK1.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

“…1), consistent with the IP-free crystal structure of IPK1 that revealed localized destabilization of the region surrounding Arg-130, which interacts with the 1-phosphate (19). We recently investigated specific roles of phosphate groups in binding and activation of IPK1, and we determined that the 5-and 6-phosphates were important for binding of the IP, whereas the 1-and 3-phosphates were important for activation of IPK1 (20). In our current study, we demonstrated that the overall stability of IPK1 is dependent on the N-lobe binding 1-and 3-phosphates (Fig.…”

“…We have also found that the 1-and 3-phosphates of the IP provide increased stability to IPK1, whereas the 5-and 6-phosphates do not affect the overall stability of IPK1, consistent with an unstable N-lobe and a stable C-lobe in the absence of IP. Moreover, previous kinetic studies of IPK1 link substrate specificity to kinase activation (20). We contend that IPK1 stability is linked to IPK1 activation because IPK1 stability correlates with the active state in previous crystal structures (19), IPs that display low IPK1 kinase activity (k cat ) correlate with a decreased ability to stabilize IPK1 (20), and kinase stability has been validated to be a precursor for activation of PKs, for which IPK1 shares similar features (9,15).…”

“…Wild-type IPK1 data were previously obtained and are provided for comparison (20 as compared with the structure of wild-type IPK1 (Fig. 5A).…”

“…We created double cysteine mutants of IPK1 at different sites near residues 110 - 140, the region of the N-lobe that was previously observed to be unstable in the absence of IP (19). We tested the kinase activity of these mutants using its physiological substrate, IP 5 , and 3,4,5,6-IP 4 , a poor IPK1 substrate (20), in reducing and nonreducing conditions (Fig. 3).…”

“…The first crystal structures of IPK1 revealed how IP 5 is phosphorylated on the 2Ј-hydroxyl of the inositol ring to yield IP 6 , but the recognition of all five phosphate groups and the axial hydroxyl group did not offer explanations for how it excludes IPs with fewer than five phosphates from its pool of potential substrates (1,15). For PKs, the stability of the N-and C-lobes is a critical precursor for kinase activation (16 -18), and these IPK1 structures indicated that a similar mechanism might be employed for some IPKs (20).…”