The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch

Abstract

PH domains, by binding to phosphoinositides, often serve as membrane-targeting modules. Using crystallographic, biochemical, and cell biological approaches, we have uncovered a mechanism that the integrin-signaling adaptor Skap-hom uses to mediate cytoskeletal interactions. Skap-hom is a homodimer containing an N-terminal four-helix bundle dimerization domain, against which its two PH domains pack in a conformation incompatible with phosphoinositide binding. The isolated PH domains bind PI[3,4,5]P(3), and mutations targeting the dimerization domain or the PH domain's PI[3,4,5]P(3)-binding pocket prevent Skap-hom localization to ruffles. Targeting is retained when the PH domain is deleted or by combined mutation of the PI[3,4,5]P(3)-binding pocket and the PH/dimerization domain interface. Thus, the dimerization and PH domain form a PI[3,4,5]P(3)-responsive molecular switch that controls Skap-hom function.

Figure 1

Structure of Skap-hom. (A) Schematic showing Skap-hom dimerization (DM), PH and SH3 domains, and two intervening linkers (L). Potential tyrosine phosphorylation (Y) sites are indicated, and point mutations studied herein are indicated below. The domain structure of the Skap-hom ΔPH mutant is also shown. (B) Structure-based sequence alignment of the Skap-hom and Skap55 N-termini and selected PH domains. Identical residues are shaded red, highly conserved residues are in red type. Residue numbering and secondary structure for mouse Skap-hom are indicated above the alignment. DM and PH domain borders are delineated by brackets. Dashed lines indicate disordered regions in the crystal structure. Blue triangles below the alignment mark Skap-hom residues involved in the dimer interface, black ovals indicate residues in the interface between the DM and PH domains, and black squares mark PH domain residues implicated in lipid head group interaction. PDB entries 1U5D (Skap55), 1H10 (Akt1), 1FAO (Dapp1), 1FGY (Grp1), 1BTK (Btk), and 1W1D (Pdk-1) were used to align PH domain structures. (C) Ribbon diagram of the dimeric DM-PH structure of Skap-hom. The view in the upper panel is along the approximate 2-fold axis of symmetry of the four-helical bundle (top view) and the lower panel is a perpendicular view (front view). The two PH domains are shown in red and yellow, the dimerization domain of one subunit is shown in blue and the other in green. The side chains of Lys125, Arg140 and Tyr151 are shown in stick form to mark the location of the phosphoinositide binding pocket (see Figure 3C).

Figure 2

Interface between the Skap-hom PH and DM domains. (A) The PH domain is shown in red and the DM domain in blue and green as in Figure 1C. Salt-bridges are indicated by dashed lines. Note the helical conformation of the β1-β2 loop (residues Arg127 – Phe135) and the insertion of PH domain residues Phe132 and Leu133 into a hydrophobic pocket on the DM domain. (B) Stereo representation of the DM domain showing the side chains of all residues, with salt-bridges highlighted. Representative charged residues are labeled. (C) View of one subunit of the DM domain showing the hydrophobic and charged residues in the dimerization interface. (D) Superposition of the Skap-hom DM (green and blue) and the dimerization domain of Nuclear Export Protein Nep/Ns2 of Influenza A (magenta) (Akarsu et al., 2003), which shares a similar overall topology but no clear sequence relationship.

Figure 3

Comparison of the isolated Skap-hom PH and DM-PH structures. (A) Ribbon diagram showing one of the PH domains in the DM-PH structure. (B) Isolated Skap-hom PH domain. Note the marked difference in conformation of the β1–β2 loop. The helical conformation (α3) seen in the DM-PH structure is incompatible with phosphoinositide binding (see text). Superposition of the Skap-hom PH domain (blue) shown in (C) with the Akt PH domain (magenta) complexed with Ins[1,3,4,5]P₄. Akt residues involved in phosphoinositide binding are labeled in bold italic type; the corresponding residues in Skap-hom are in regular type. (C) Electrostatic surface representation of the dimeric DM-PH structure and close-up of one lipid-binding pocket. The highly basic surfaces (shaded blue) of the phosphoinositide binding pockets are on opposite faces of the dimer and are recessed relative to the DM domain. The expected position of the head group is modeled into one of the PH domains (inset), based on superposition of the Akt PH domain complexed with Ins[1,3,4,5]P₄. Note the obvious steric clash with the head group due to the helical conformation of the β1–β2 loop.

Figure 4

Skap-hom can homodimerize. (A) Full-length Skap-hom constructs, epitope-tagged with either Flag or HA, were expressed in 293T cells. Total cell lysates (TCL) and anti-HA immunoprecipitates (IP) probed with anti-HA (upper) or anti-Flag (lower) antibodies are shown. The diffuse band present in the anti-HA blots is the 12CA5 heavy chain used for immune precipitation. (B) HA-tagged full-length Skap-hom was expressed with either wild type (WT) or a dimerization domain mutant (DM*) DM-PH fragment and immunoprecipitated with anti-HA antibodies. The resulting immunoblot was probed with anti-Skap-hom antibodies. The upper bands are HA-Skap-hom and the lower bands are the DM-PH, respectively.

Figure 5

Phosphoinositide binding of the Skap-Hom PH and DM-PH fragments.. (A) Dot blot assays of Skap-hom PH and DM-PH fragments with the indicated phosphoinositides. (B) Florescence polarization-based binding assays of the isolated Skap-hom PH domain and its R140M mutant to PI[3,4,5]P₃. (C) Florescence polarization-based binding assays of the WT Skap-hom DM-PH fragment and its mutants (D129K, D129A, K56A, R140M) with PI[3,4,5]P₃.

Figure 6

Localization of GFP-tagged Skap-hom variants in Skap-hom^−/− bone marrow-derived macrophages (BMM). Skap-hom^−/− BMM were reconstituted with (A) GFP-tagged WT Skap-hom, (B) GFP alone, or (C–G) the indicated GFP-tagged variants of Skap-hom. (H–N) Cells were fixed and counter-stained with rhodamine-labeled phalloidin to visualize the actin cytoskeleton. (O–U) Merged GFP and Rhodamine channels for each infection. WT Skap-hom (A, H and O) co-localizes with actin-rich ruffles. The DM* dimerization domain triple mutant (panels C, J and Q) and the R140M PH domain mutant (panels D, K and R) are diffusely localized. Deletion of the PH domain (ΔPH; E, L and S) results in a protein that localizes to actin ruffles. Skap-hom bearing both the R140M and either the D129K (D129K:R140M panels F, M, T) or the K56A (K56A:R140M panels G, N and U) mutations co-localize with actin-ruffles.