Resolution of structure of PIP5K1A reveals molecular mechanism for its regulation by dimerization and dishevelled

Abstract

Type I phosphatidylinositol phosphate kinase (PIP5K1) phosphorylates the head group of phosphatidylinositol 4-phosphate (PtdIns4P) to generate PtdIns4,5P2, which plays important roles in a wide range of cellular functions including Wnt signalling. However, the lack of its structural information has hindered the understanding of its regulation. Here we report the crystal structure of the catalytic domain of zebrafish PIP5K1A at 3.3 Å resolution. This molecule forms a side-to-side dimer. Mutagenesis study of PIP5K1A reveals two adjacent interfaces for the dimerization and interaction with the DIX domain of the Wnt signalling molecule dishevelled. Although these interfaces are located distally to the catalytic/substrate-binding site, binding to these interfaces either through dimerization or the interaction with DIX stimulates PIP5K1 catalytic activity. DIX binding additionally enhances PIP5K1 substrate binding. Thus, this study elucidates regulatory mechanisms for this lipid kinase and provides a paradigm for the understanding of PIP5K1 regulation by their interacting molecules.

Figure 1. PIP5K1A structure.

(a,b) Crystal structure of zebrafish PIP5K1A kinase catalytic domain and its comparison with that of PKA. The N- and C-terminal segments outside the kinase core domain are omitted to better illustrate the protein kinase fold. In both panels, the N-lobe is coloured grey and the C-lobe is yellow. The ‘C helix' is highlighted in red. The substructure harbouring the lipid kinase's DLKGS motif is highlighted in blue. The N-terminal secondary structures found only in phosphatidylinositol phosphate kinases are shown in pink. The structure C-terminal to PKA's αF, missing in the phosphatidylinositol phosphate kinases, is coloured green. The disordered regions are indicated by dotted lines. (c,d) The DLKGS motifs of zPIP5K1A and PIP4K2B. The substructure (blue) harbouring the DLKGS motif reveals differences between zPIP5K1A (c) PIP4K2B (d). The C-lobe of the kinase is coloured yellow. Dashed lines represent potential hydrogen bonds.

Figure 2. Dimerization of PIP5K1A catalytic domain.

(a) SEC/MALLS shows that zPIP5K1A is dimeric in the solution. dRI, differential refractive index (blue trace). The calculated molecular weight is indicated by the red dots. (b) The side-to-side dimerization of zPIP5K1A. (c) A detailed view of the interactions that stabilize the dimer. This image corresponds approximately to the same view as that in b. (d) The head-to-head dimerization of PIP4K2B (PDB: 1BO1). (e) SEC elution profiles of the wild-type zPIP5K1A protein (blue) and its monomeric mutant DLV (brown). (f) Basal lipid kinase activity of the wild-type zPIP5K1A protein and its monomeric mutants. D84R, D84R mutant protein; PIP₂, PtdIns4,5P₂; R287D, D84R/R287D mutant protein; WT, wild-type protein. (g) Prevention of zPIP5K1A head-to-head dimerization by the β1–β2 loop. zPIP5K1A (cyan) is superimposed to one protomer of a PIP4K2A dimer (yellow, on the left). The β1–β2 loop of zPIP5K1A (blue) folds back, which is different from the fold of the corresponding region in PIP4K2B (orange), blocking dimerization in a head-to-head manner observed in PIP4K2B. The other protomer of the PIP4K2B dimer is shown in pink (right). (h) The phylogenetic relationship of type I (PIP5K), II (PIP4K) and III (PIKfyve) kinases. A multiple sequence alignment shows the conservation of residues at the dimeric interfaces of PIP5K (red) and PIP4K (blue). At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Rc, Ricinus communis; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. (i) Hydrogen bonds between the two α1 helices help to stabilize the PIP4K2B dimer.

Figure 3. PIP5K1A interface for DIX interaction.

(a,b) Schematic representation of mutated zPIP5K1A residues. Green marks those residues, when mutated, causing insolubility. Deep red and blue mark those critical for DIX binding. The rest do not affect the binding. (b) 90° Rotation of a with the membrane interaction interface at the back. (c) Interaction of recombinant zPIP5K1A and its mutants with recombinant DIX. GST-pull-down was performed with His-tagged zPIP5K1A proteins and GST-tagged DIX with GST as a control. The proteins were detected by western blotting. WT, wild type.

Figure 4. Regulation of PIP5K1A1 by DIX.

(a) Regulation of the lipid kinase activity of zPIP5K1A by DIX. In vitro lipid kinase assay was performed using recombinant zPIP5K1A or its mutant proteins in the presence or absence of recombinant DIX proteins. Phospholipids were separated on thin-layer chromatography (TLC) plates (a representative TLC image is shown) and quantified by a phosphoimager. Dvl-induced lipid kinase activity of wild-type (WT) PIP5K1a was taken as 100%. Error bars stands for standard errors. The assays were repeated at least twice. (b) Expression of wild-type, but not E129A, zPIP5K1A rescues the inhibition of Wnt3a-induced reporter gene activity by PIP5K1A knockdown in HEK293 cells. Data are presented as means±s.d. (Student's t-test, n=3). Protein expression was examined by western analysis.

Figure 5. Regulation of zPIP5K1A enzymatic activity.

(a) Binding of zPIP5K1A (wild type (WT)) and its monomeric DLV mutant to PtdIns4P-containing liposomes measured by using a liposome floatation assay in the presence of DIX or GST. (b) DIX stimulates PIP5K1 binding only to liposomes containing PtdIns4P. The liposome floatation assay was performed using phosphatidylcholine/phosphatidylserine liposomes with or without PtdIns4P. (c) Effect of the E129A and Q194A mutations on Dvl-induced binding of zPIP5K1A to PtdIns4P-containing liposomes. (d) Effects of DIX on the kinase activity of zPIP5K1A (WT) and its monomeric DLV mutant. Data are presented as means±s.d. (e) Interaction of zPIP5K1A (WT) and its monomeric DLV mutant with DIX. GST-pull-down was performed with His-tagged zPIP5K1A proteins and GST-tagged DIX with GST as a control. The proteins were detected by western blotting. (f) Assessment of the binding affinities of zPIP5K1A (WT) and its monomeric DLV mutant for DIX using ITC. The experiments were repeated twice times. The results from one of the experiments are shown.