Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P₂ metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P₂ synthesis during clathrin-mediated SV endocytosis.

Introduction

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)⁵ plays a key role in a variety of cell physiological pathways including but not limited to CME, neurotransmitter release (31) and SV cycling, phagocytosis, cell signaling and proliferation, and regulation of the actin cytoskeleton, as well as nuclear functions (1–7). In mammals these diverse roles of PI(4,5)P₂ are regulated by only three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) isozymes. Although PIPKIα and -β are ubiquitous proteins, the type Iγ isozyme is most highly expressed in the brain where it localizes to synaptic sites (8). PIPKIγ is also present at focal adhesion sites in non-neuronal cells (9, 10).

An overwhelming body of data implicates PI(4,5)P₂ in CME and in the exo-/endocytic cycling of SV membranes (2–4). Recruitment of clathrin adaptors, including epsins, CALM/AP180, Dab2, HIP1/1R, and AP-2, to the plasma membrane strictly requires the presence of PI(4,5)P₂, which associates directly with cognate recognition motifs and domains within these proteins. AP-2 is a heterotetrameric complex composed of α, β2, μ2, and σ2 subunits that fold into a largely α-helical solenoid (termed the “core”) with two appendage domains joined to the core by long flexible linkers (11). Membrane recruitment of AP-2 is thought to be accomplished by a coincidence detection system involving transmembrane cargo proteins (12) bound to AP-2μ or AP-σ2 (13) via sorting motif peptides and two binding sites for PI(4,5)P₂ within the AP-2 core (11). One site is located within the N-terminal portion of the α subunit; this site is mainly responsible for targeting AP-2 to the plasma membrane (14). The second site within the μ2 subunit (15) presumably requires prior activation of AP-2 through a phosphorylation-induced conformational change by which the C-terminal sorting signal binding domain of AP-2μ gets dislodged from the AP-2 core. This conformational opening enables μ2 to recognize YXXØ-based sorting motifs within the cytoplasmic domain of transmembrane cargo proteins and to interact with PI(4,5)P₂ in the membrane (16). Hence, PI(4,5)P₂ is required for the initial targeting of AP-2 to the plasma membrane as well as for cargo recognition, which in turn serves to stabilize nascent coated pits during CME (12). Given that CME is a pathway active in most cell types, one might expect that PIPKI-mediated synthesis of PI(4,5)P₂ would be subject to common mechanisms of spatiotemporal regulation. In support of this idea, it has been reported that PIPKI isozymes associate with AP-2 via direct interaction between the kinase core and a site within AP-2μ distinct from that involved in recognizing YXXØ-based sorting motifs. Moreover, cargo protein binding has been shown to stimulate PIPKI activity, suggesting that synthesis of an endocytic pool of PI(4,5)P₂ is under the control of a positive feedback loop (17). Such a mechanism might be important for sustaining endocytosis under conditions of intense competition for PI(4,5)P₂ by other pathways.

Synapses face special challenges with respect to membrane trafficking because of the spatial and temporal constraints of chemical neurotransmission (4). A number of independent lines of evidence indicate a central role for PI(4,5)P₂ metabolism in exo-/endocytic cycling of SVs. Perturbation of PI(4,5)P₂ breakdown by genetic or acute manipulation of the inositol phosphatase synaptojanin impairs SV endocytosis at the stage of vesicle uncoating, resulting in synaptic depression following intense stimulation (18, 19). Most importantly, in mice the loss of PIPKIγ, the major synthesizing enzyme at synapses, produces defects in SV recycling including significantly reduced frequencies of spontaneous miniature postsynaptic currents and decreased rates of clathrin-mediated SV endocytosis (20). These data suggest a crucial role for PIPKIγ-mediated PI(4,5)P₂ generation in CME of SV proteins. Consistent with these results, it has been reported that the tail domain of PIPKIγ-p90, the major splice isoform in neurons, can associate directly with the β2 appendage domain (21) and/or the sorting signal binding domain of the μ2 subunit of AP-2 (22). In agreement with this, overexpression of the AP-2-binding PIPKIγ tail domain has been shown to impair depolarization-induced SV endocytosis (21). The precise structural basis of these effects, however, has remained elusive.

In this work we report on the crystal structures of a peptide derived from the 28-amino acid splice insert of the PIPKIγ-p90 tail in complex with the β2 appendage or the C-terminal sorting signal binding domain of AP-2μ. Our biochemical and structure-based mutagenesis analysis revealed that association of the natively unfolded tail domain of PIPKIγ-p90 with AP-2 involves both of these interaction sites. Moreover, the ability of the PIPKIγ tail to impair endocytic retrieval of SV proteins in primary neurons is critically dependent on its association with both AP-2β and AP-2μ. Our data indicate that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzyme activity, suggesting a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P₂ synthesis during SV endocytosis.

EXPERIMENTAL PROCEDURES

Antibodies

The monoclonal antibodies used were: anti-AP-2α_A (clone 8) or AP-2μ (clone 31) from BD Transduction Laboratories (San Jose, CA); anti-actin (C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-talin 1 (clone 8d4) and anti-HA antibodies from Sigma; anti-His₆ from Novagen (Madison, WI). Anti-AP-1/2 β1/β2 antibodies were a kind gift of Dr. Tom Kirchhausen (Harvard Medical School, Boston), and monoclonal antibodies recognizing clathrin heavy chain (TD1) and AP-2α_A (AP6) have been described previously. Polyclonal antibodies specific for PIPKIγ were raised in rabbits using amino acids 451–668 of its tail domain as an antigen and were affinity-purified.

DNA Constructs

Fusion proteins encoding GST-PIPKIγ-p90-(451–668) or the GST-β2 appendage (WT or Y815A) have been described previously (17, 23). Site-directed mutants of PIPKIγ-p90 or GST-β2-ear were generated using overlap extension PCR with mutagenic primers followed by digestion and ligation into EcoRI/NotI-cleaved pGEX4T-1. The identities of the DNA inserts and the presence of mutations were confirmed by double-stranded DNA sequencing. Constructs encoding His₆-C-μ2 or HA-PIPKIγ-p90 have been described previously (17).

Protein Purification

Constructs in pGEX4T-1 and pET28 were transformed into Escherichia coli BL21-Codon Plus^TM (DE3)-RP competent cells (Stratagene). His₆- or GST-tagged fusion proteins were expressed and purified using HIS-Select^TM nickel affinity gel (Sigma) or GST Bind® resin (Novagen) following the manufacturer's instructions. For isothermal titration calorimetry (ITC) experiments, the His₆-C-μ2 or His₆-β2 appendage was purified using HisTrap HP columns (GE Healthcare) according to the manufacturer's instructions. The proteins were further purified by size exclusion chromatography (Superdex S200, Amersham Biosciences) in 20 mm Hepes, pH 7.4, containing 200 mm NaCl.

Preparation of Rat Brain and Synaptosomal Extracts

Rat brain extract was prepared from frozen rat brains homogenized in 10 ml of homogenization buffer (4 mm Hepes, pH 7.4, 320 mm sucrose, 1 mm PMSF, and protease inhibitor mixture (Sigma)) by using a glass-Teflon homogenizer. The homogenate was centrifuged for 10 min at 1000 × g. The supernatant was then adjusted to 20 mm Hepes, pH 7.4, 100 mm NaCl, and 2 mm MgCl₂ containing 1% Triton X-100. Following incubation for 10 min on ice, the extract was centrifuged for 15 min at 43,500 × g. The resulting supernatant was precleared by ultracentrifugation for 15 min at 184,000 × g and used for pulldown experiments. For preparation of rat brain synaptosomal extracts, homogenized rat brain was centrifuged for 10 min at 1000 × g. Postnuclear supernatant was centrifuged for 20 min at 14,600 × g. The crude synaptosomal pellet was resuspended in 10 ml of homogenization buffer containing 1 mm PMSF and protease inhibitor mixture followed by further centrifugation for 20 min at 14,600 × g. The resulting synaptosomal pellet was resuspended in homogenization buffer containing 1 mm PMSF and protease inhibitor mixture and adjusted to 20 mm Hepes, pH 7.4, 100 mm NaCl, and 2 mm MgCl₂ containing 0.5% CHAPS. Synaptosomes were incubated for 40 min at 4 °C. The resulting lysate was precleared by further centrifugation steps, first for 15 min at 35,000 × g and then for 15 min at 184,000 × g, and used for pulldown experiments.

Affinity Chromatography

For GST pulldowns GST or GST-PIPKIγ-p90-(451–668) was incubated with 3 mg of rat brain extract (in 20 mm Hepes, pH 7.4, 320 mm sucrose, 100 mm NaCl, 2 mm MgCl₂, 1% Triton X-100, 1 mm PMSF, and protease inhibitor mixture) or 900 μg of rat brain synaptosomal extract (in 20 mm Hepes, pH 7.4, 320 mm sucrose, 100 mm NaCl, 2 mm MgCl₂, 0.5% CHAPS, 1 mm PMSF, and protease inhibitor mixture) for 1 h by end-over-end rotation at 4 °C. The beads were extensively washed four times, resolved by SDS-PAGE, and analyzed by immunoblotting. Alternatively, 7 μg of GST or GST-PIPKIγ-p90-(451–668) was incubated with 7 μg of His₆-talin FERM domain or His₆-C-μ2 for 1 h at 4 °C in 100 μl of pulldown buffer (20 mm Tris/HCl, pH 7.4, 150 mm NaCl, 5 mm imidazole, 2 mm MgCl₂, and 1% Triton X-100). For β2-ear pulldowns, 50 μg of GST or GST-PIPKIγ-p90-(451–668) was incubated with an equimolar amount of His₆-β2-ear for 1 h at 4 °C in 500 μl of pulldown buffer (20 mm Tris/HCl, pH 7.4, 150 mm NaCl, 5 mm imidazole, 2 mm MgCl₂, and 0.05% Tween 20). Beads were washed extensively (four times), resolved by SDS-PAGE, and analyzed by immunoblotting.

Crystallography of the β2 Appendage/PIPKIγ-p90 Peptide Interaction

An E. coli construct containing the gene coding for the ear domain of the β2 subunit of AP-2 (His₆-β2 appendage, amino acids 705–937) was cultured in “double” LB medium supplemented with kanamycin (40 μg/ml each). When the absorbance at 600 nm reached 0.7, overexpression was induced by supplementing the culture with 0.8 mm isopropyl 1-thio-β-d-galactopyranoside, and cells were harvested 3 h later. His₆-β2ear was isolated by nickel-nitrilotriacetic acid affinity chromatography (Qiagen), and the His₆ tag cleaved off by thrombin and β2-ear was purified by size exclusion chromatography (Superdex S75, Amersham Biosciences). The purified protein was dialyzed against 20 mm Tris, pH 8, and 150 mm NaCl, concentrated to about 60–80 mg/ml, and supplied with a 5-fold molar surplus of the PIPKIγ-p90 pentadecapeptide YFPTDERSWVYSPLH. Crystals were grown at 18 °C using the sitting drop vapor diffusion method in the presence of trimethylphenylammonium-exchanged hectorite (<2 μm). The reservoir solution contained 18% polyethylene glycol 8000, 100 mm Hepes, pH 7.5, and 4 mm dithiothreitol. Drops were prepared by mixing 1 μl of reservoir and 1 μl of protein/peptide solution at 80 mg/ml. Crystal showers with poorly defined microcrystals not suitable for single crystal x-ray studies appeared within 10–30 s. To slow the crystallization process, nucleation seeds were introduced in the form of trimethylphenylammonium-exchanged hectorite (<2 μm). Thin plate-like crystals formed around these sites within 60 min and reached their final size in about 3 h at 18 °C.

Single crystals of the AP-2 β2 ear co-crystallized with the above mentioned PIPKIγ pentadecapeptide were soaked briefly in a cryoprotection medium of 18% polyethylene glycol 8000, 100 mm Hepes, pH 7.5, 4 mm dithiothreitol, and 15% v/v glycerol, mounted in a nylon loop, and then flash-cooled in liquid N₂. X-ray data were collected at Beamline BL2 at BESSY-II, Berlin, and processed using HKL2000 (24) and Scalepack. The space group was determined as P2₁2₁2₁ with a solvent content of 51%, indicating 1 molecule/asymmetric unit. The unit-cell parameters were a = 37.56, b = 83.47, and c = 91.60 Å. The crystals diffracted to 1.83 Å resolution. The data set was 95% complete with an overall R_merge of 6.0%. The phase problem was solved by molecular replacement with the CCP4 (25) program MOLREP using the 1.60 Å structure of the β2 ear (Protein Data Bank code 2G30) as a model (26). All water molecules and ligand atoms were omitted from the starting model. Subsequent cycles of the isotropic B-value and positional refinement to 1.83 Å resolution were performed using Refmac5 (27). The peptide chain and missing residues were built manually using the model-building program Coot (28). Orientation of the amino acid side chains and bound water molecules was modeled based on electron density < 3σ in F_o − F_c Fourier maps. Modeled water molecules that refined with electron density > 3σ were deleted. The final R-factor for the resolution range of 60 to 1.83 Å was 19.3% (R_free 23.6%). The Ramachandran plot performed with PROCHECK indicated that 93.6% of the residues fell within the sterically most favored region with an additional 6.4 and 0% within the allowed and generously allowed regions, respectively. The statistics of the resulting structure (structural data available under Protein Data Bank codes 3H85 and 3H1Z) are reported in Tables 1–3.

TABLE 1.

Data collection and refinement statistics

	AP-2(βear) + PIPKIγ-p90 peptide
Data collection
Space group	P212121
Cell dimensions
a, b, c (Å)	37.56, 83.47, 91.60
α, β, γ (°)	90.00, 90.00, 90.00
Resolution (Å)	50-1.83
Rsym or Rmerge	0.060 (0.461)a
I/σI	20.38 (1.90)a
Completeness (%)	94.6 (88.1)a
Redundancy	4.5 (3.6)a
Refinement
Resolution (Å)	60-1.83
No. reflections	24,729
Rwork/Rfree	19.3/23.6
No. atoms
Protein	1,881
Ligand	136
Water	253
B-Factors
Protein	22.5
Ligand	23.3
Water	33.1
r.m.s.b deviations
Bond lengths (Å)	0.016
Bond angles (°)	1.618

^a The highest resolution shell is shown in parentheses.

^b r.m.s., root mean square.

TABLE 2.

AP-2β-appendage domain in complex with PIPKIγ-p90-derived peptide YFPTDERSWVYSPLH, hydrogen bonds/salt bridges, and buried surface area (BSA) (see Fig. 2B)

Peptide		Protein interface
Residue	Atom	Residue	Atom	Distance	BSA
				Å2	Å2
Tyr-639					53.80
Phe-640					138.59
Pro-641					2.32
Thr-642					29.02
Asp-643	N	Gln-756	OE1	3.74	2.75
Asp-643	O	Gln-756	NE2	2.94
Glu-644	OE1	Lys-808	NZ	3.95	4.79
Arg-645
Ser-646					26.29
Trp-647					119.93
Val-648	N	Ile-755	O	2.79	106.97
Val-648	O	Ile-755	N	3.17
Tyr-649	OH	Lys-808	NZ	3.02	52.56
Ser-650	N	Phe-753	O	3.02	82.98
Ser-650	OG	Leu-770	O	2.67
Pro-651	O	His-773	ND1	3.00	42.92
Leu-652	N				66.10
His-653	N	His-773	O	2.74	62.43
His-653	O	Thr-774	OG1	3.39
Total				11

TABLE 3.

AP-2β-appendage domain in complex with Eps15-derived peptide SFGDGFADF, hydrogen bonds/salt bridges, and buried surface area (BSA)

Peptide		Protein interface I (chain A)
Residue	Atom	Residue	Atom	Distance	BSA
				Å2	Å2
Ser-1					3.43
Phe-2					145.66
Gly-3					13.92
Asp-4	OD1
Asp-4	OD1
Gly-5	N	Tyr-815	OH	2.80	23.06
Gly-5	O	Tyr-815	OH	3.82
Phe-6					67.26
Ala-7
Asp-8	OD2	Tyr-815	OH	2.56	30.54
Phe-9					144.94

Crystallography of the μ2/PIPKIγ-p90 Peptide Interaction

An E. coli construct containing the gene coding for the C-terminal domain of the μ2 subunit of AP-2 (His₆-C-μ2; amino acids 158–435) was cultured in double LB medium supplemented with kanamycin (40 μg/ml each). When A_{600 nm} reached 0.7, overexpression was induced by supplementing the culture with 0.8 mm isopropyl 1-thio-β-d-galactopyranoside, and cells were harvested 4 h later. His₆-C-μ2 was isolated by nickel-nitrilotriacetic acid affinity chromatography (Qiagen), and the His₆ tag cleaved off by thrombin and C-μ2 was purified by size exclusion chromatography (Superdex S200, Amersham Biosciences). The purified protein was dialyzed against 10 mm Hepes, pH 7.5, 150 mm NaCl, and 4 mm dithiothreitol, concentrated to about 22 mg/ml, and supplied with a 5-fold molar surplus of the human PIPKIγ octapeptide SWVYSPLH. Crystals were grown at 18 °C using the sitting drop vapor diffusion method. The reservoir solution contained 1.4 m sodium formate, 50 mm NiCl, and 0.1 m sodium acetate, pH 6.0, and drops were prepared by mixing 1 μl of reservoir and 1 μl of protein/peptide solution at 22 mg/ml. Large hexagonal rod-like crystals formed within 24 h and reached their final size in 2–3 days at 18 °C. Optically flawless crystals were soaked briefly in a cryoprotection medium of 1.4 m sodium formate, 50 mm NiCl₂, 0.1 m sodium acetate, pH 6.0, and 15% (v/v) glycerol, mounted in a nylon loop, and then flash-cooled in liquid N₂. X-ray data were collected at Beamline BL1 at BESSY-II, Berlin, and processed using HKL2000 (24) and Scalepack. The space group was determined as P6₄ with a solvent content of 74%, resulting in an occupancy of 1 molecule/asymmetric unit. The unit-cell parameters were a = b = 125.30 and c = 74.55 Å. The crystals diffracted to 2.60 Å resolution, and the data set was 93% complete with an overall R_merge of 6.9%. The phase problem was solved by molecular replacement with CCP4 program MOLREP (Collaborative Computational Project, 1994) using the 2.65 Å structure of the μ2 adaptin subunit (Protein Data Bank code 1BW8) as a model (29). All water molecules and ligand atoms were omitted from the starting model. After rigid body refinement the R-value was 33.4% (R_free = 37.1%) for data between 40 and 2.8 Å resolution. Subsequent cycles of isotropic B-value and positional refinement to 2.60 Å resolution were performed using Refmac5 (27). The peptide chain and missing residues were built manually using the model building program Coot (28). The orientation of the amino acid side chains and bound water molecules were modeled based on electron density < 3σ in F_o − F_c Fourier maps, and water molecules that refined with B-values > 3σ were deleted. The final R-factor for the resolution range, 41 to 2.60 Å, was 23.3% (R_free 26.3%). The Ramachandran plot performed with PROCHECK indicated that 84.3% of the residues fell within the sterically most favored region with an additional 15.2 and 0.4% within the allowed and generously allowed regions, respectively. The statistics of the resulting structure (structural data available under Protein Data Bank codes 3H85 and 3H1Z) are reported in Tables 4–6. Figs. 1 and 3 were drawn with PyMOL.

TABLE 4.

Data collection and refinement statistics

	AP-2μ + PIPKIγ-p90 peptide
Data collection
Space group	P64
a, b, c (Å)	125.30, 125.30, 74.55
α, β, γ (°)	90.00, 90.00, 120.00
Resolution (Å)	50-2.60
Rsym or Rmerge	0.069 (0.484)a
I/σI	15.96 (1.34)a
Completeness (%)	92.6 (49.0)a
Redundancy	3.5 (1.8)a
Refinement
Resolution (Å)	41.01-2.60
No. reflections	18,278
Rwork/Rfree	23.3/26.3
No. atoms
Protein	2,039
Ligand	71
Water	33
B-Factors
Protein	64.6
Ligand	64.3
Water	69.6
r.m.s.b deviations
Bond lengths (Å)	0.008
Bond angles (°)	1.151

^a Highest resolution shell is shown in parentheses.

^b r.m.s., root mean square.

TABLE 5.

AP-2μ-(158–435) in complex with PIPKIγ-p90-derived peptide SWVYSPLH, hydrogen bonds/salt bridges, and buried surface area (BSA) (see Fig. 4B)

Peptide		Protein interface I
Residue	Atom	Residue	Atom	Distance	BSA
				Å2	Å2
Ser-646	N	Glu-391	OE1	3.43	87.74
Ser-646	N	Glu-391	OE2	3.31
Trp-647	O	Arg-423	NH1	2.73	179.53
Val-648					6.96
Tyr-649	OH	Asp-176	OD1	2.65	137.40
Tyr-649	OH	Lys-203	NZ	2.65
Tyr-649	OH	Arg-423	NH2	3.40
Tyr-649	OH	Arg-423	NE	3.16
Ser-650	N	Val-422	O	2.93	38.78
Ser-650	O	Val-422	N	2.82
Pro-651					29.96
Leu-652	N	Lys-420	O	2.87	137.87
His-653	NE2	Arg-402	O	3.25	45.78

TABLE 6.

AP-2μ-(158–435) in complex with GABA_A receptor γ2-derived peptide DEEYGYECLD, hydrogen bonds/salt bridges, and buried surface area (BSA)

Peptide		Protein interface I
Residue	Atom	Residue	Atom	Distance	BSA
				Å2	Å2
Asp-1					7.62
Glu-2	OE2	Lys-319	NZ	2.94	43.56
Glu-3
Tyr-4	OH	Gln-318	NE2	3.78	131.47
Gly-5					17.21
Tyr-6	OH	Asp-176	OD1	2.53	136.77
Tyr-6	OH	Lys-203	NZ	2.69
Glu-7	N	Val-422	O	2.90	40.02
Glu-7	O	Val-422	N	2.84
Cys-8					26.93
Leu-9	N	Lys-420	O	2.84	149.11
Asp-10					33.08

FIGURE 1.

Crystal structure of the β2 appendage domain complexed with a PIPKIγ-p90-derived peptide. A, ribbon diagram showing the binding site for the PIPKIγ-p90-derived peptide (corresponding to amino acids 639–653 (blue)) in complex with the β2 appendage (gray). The peptide binds to an elongated pocket at the side site of the β2 appendage sandwich subdomain. B, close-up view showing direct molecular contacts between the PIPKIγ-p90 peptide (blue) and the side site of the β2 appendage. Phe⁶⁴⁰ is keyed into the hydrophobic pocket (shown as spheres) formed by Ala-806, Tyr-815, Gln-804, and Asn-758 of β2. The entire sequence of the bound peptide participates in a β-sheet-like hydrogen bonding with β2. C, surface representation of the PIPKIγ-p90 peptide binding interface with the β2 appendage including an overlay with the previously characterized Eps15-derived peptide, SFGDGFADF (yellow), (23) to compare the binding of the two peptides.

FIGURE 3.

Crystal structure of AP-2μ-(158–435 (gray) complexed with the PIPKIγ-p90-derived peptide (blue). A, ribbon diagram showing the binding site for the PIPKIγ-p90-derived peptide (corresponding to amino acids 646–653 (blue)) in complex with the sorting signal binding domain of AP-2μ (gray). The peptide binds to a banana-shaped pocket on the AP-2μ surface used for other YXXØ motif-containing cargo. B, close-up view showing direct molecular contacts between the PIPKIγ-p90 peptide and AP-2μ-(158–435). Trp-647 is located within a hydrophobic pocket formed by Glu-391, Gln-318, and Pro-393, thereby providing a third specificity determinant within the PIPKIγ-p90 peptide for AP-2μ. C, surface representation of the PIPKIγ-p90 peptide binding interface with AP-2μ, including an overlay with the previously characterized GABA_A receptor γ2 subunit-derived peptide, DEEYGYECLD (residues 362–371 (yellow) (42)), to compare binding of the two peptides.

Isothermal Titration Calorimetry

High sensitivity ITC was performed on a VP-ITC device (MicroCal) at 25 °C using gel-filtrated protein at concentrations of 60 to 100 μm in buffer (20 mm Hepes, pH 7.4, and 200 mm NaCl). The PIPKIγ-p90 peptide (YFPTDERSWVYSPLH) was dialyzed against this buffer and injected in 30 steps of 10 μl each from 1.12 mm stock solutions at 5-min intervals. Both the peptide and protein samples were gently degassed under stirring prior to the experiment to avoid air bubbles. Peptide was also injected into dialysis buffer to measure the heat of dilution. Base-line subtraction and peak integration were accomplished using Origin 7.0 as described by the manufacturer (MicroCal Software). All reaction heats were normalized with respect to the molar amount of peptide injected and corrected for the heat of dilution. Dissociation constants, K_D, were calculated in Origin 7.0 using nonlinear regression analysis according to the “one set of sites” model. Initial injections were always excluded from the evaluation because they usually suffer from sample loss due to mounting of the syringe and equilibration preceding the actual titration.

PIPK Activity Assays

HEK293 cell extracts were incubated with GST fusion proteins or assayed directly. Samples were incubated in kinase buffer (50 μl) containing 10 μg of phosphatidylinositol 4-phosphate, 200 μm ATP, and 10 μCi of [γ-³²P]ATP for 20 min at 37 °C. Lipids were extracted and analyzed as described previously (17).

Quantification of Synapto-pHluorin Exo-/Endocytic Cycling in Primary Hippocampal Neurons

Synapto-pHluorin (a fusion protein between pHluorin green fluorescent protein and synaptobrevin 2) partitioning between vesicular and surface-stranded plasmalemmal pools was assayed according to published procedures. Briefly, primary hippocampal neurons were prepared from Wistar rats within 24 h after birth and co-transfected with plasmids encoding for ecliptic pHluorin-tagged synaptobrevin 2 (synapto-pHluorin) and mRFP (control) or mRFP fused to the C-terminal tail of PIPKIγ-p90 comprising amino acids 451–668 (WT or indicated mutants thereof) by calcium phosphate-DNA co-precipitation on day 14 in vitro. Neurons were analyzed by live imaging analysis within 16 to 18 h after transfection using a charge-coupled device camera (AxioCam, Carl Zeiss, Inc.) on an inverted microscope (Axiovert 200M, Carl Zeiss, Inc.) with ×40 oil immersion objectives (1.4 NA; Carl Zeiss, Inc.). Images were taken at room temperature every 5 s with 200 ms excitation at 488 nm. To obtain and analyze the images, Slidebook 4.0.10 software (Intelligent Imaging Innovations, Inc.) was used. Surface and vesicle fractions of synapto-pHluorin in neurons were determined by applying physiological buffer (170 mm NaCl, 3.5 mm KCl, 0.4 mm KH₂PO₄, 20 mm TES, 5 mm NaHCO₃, 5 mm glucose, 1.2 mm Na₂SO₄, 1.2 mm MgCl₂, and 1.3 mm CaCl₂, pH 7.4) followed by acidic buffer, where TES was replaced with MES, pH 5.5, and basic buffer with 50 mm NH₄Cl.

For each neuron, more than 50 boutons were quantified. The fraction (p) of surface-stranded synapto-pHluorin (spH) was calculated by the following equation: p_surface = [spH]_surface/([spH]_surface + [spH]_vesicle) = (F_physio − F_acidic)/(F_physio − F_acidic) + (F_basic − F_physio).

RESULTS

Structural Basis for the Association of PIPKIγ-p90 with the β2 Appendage Domain of AP-2

PIPKIγ-p90 has been reported to associate with AP-2 via three distinct interaction sites: direct binding of the PIPK catalytic core to AP-2μ at a site distinct from the YXXØ motif recognition site (17) (site 1) and association of a peptide rich in aromatic and hydrophobic amino acids contained within the 28-amino acid splice insert of the p90 tail domain with either AP-2μ (22) (site 2) or the AP-2 β appendage domain (21) (site 3). At present it is unclear to what extent these sites determine AP-2·PIPKIγ-p90 complex formation and what is the precise molecular basis of these interactions. Furthermore, physiologically it is possible that multiple interaction surfaces for PIPKIγ-p90 within AP-2 are involved in the regulation of CME, i.e. during recycling of SV proteins at presynaptic nerve terminals (32).

We therefore decided to characterize these interaction surfaces in more detail, focusing on the binding of the human PIPKIγ-p90 tail to the purified β2 appendage or μ2-(158–435) (see below). Truncation analysis (data not shown) narrowed the minimal binding site required for association of the PIPKIγ-p90 tail with the β2 appendage to a 15-amino acid sequence within the 28-amino acid splice insert of p90, in complete agreement with previous work (21). The corresponding peptide was synthesized and used for co-crystallization experiments with the purified β2 appendage.

The crystals obtained diffracted to a maximal resolution of 1.83 Å, and the structure model was refined with excellent B-factors for protein and peptide ligand (Table 1). The p90 peptide is seen to accommodate the sandwich subdomain of the β2 appendage (Fig. 1 A) at a location similar to that reported previously for a short peptide fragment derived from Eps15 (23) (Fig. 1C and Tables 2 and 3). The peptide largely binds via aromatic amino acids Phe⁶⁴⁰ (residues within human PIPKIγ are indicated by superscripted numbering), Trp⁶⁴⁷, and Tyr⁶⁴⁹. Phe⁶⁴⁰, surrounded by Ala-806, Tyr-815, Gln-804, and Asn-758, exhibits the largest buried surface area (138.6 Ų; see Table 2) within β2, Trp⁶⁴⁷ projects into a shallow pocket formed by Lys-808, Gln-756, and Ala-754 (Fig. 1B). In comparison, Tyr⁶³⁹ displays a buried surface area of only 53.8 Ų and does not form hydrogen bond interactions. This explains the findings by Thieman et al. (36) that mutation of Phe⁶⁴⁰ to Ala impairs association with the AP-2β appendage, whereas Tyr⁶³⁹ → Ala does not. Additional major contacts involve Ser⁶⁵⁰, a site implicated previously in association with AP-2β by site-directed mutagenesis (21), as well as further hydrogen bonds along the entire sequence of the bound peptide resembling a β-sheet hydrogen bonding pattern (Fig. 1B and Table 2). Interestingly, Thieman et al. (36) also reported that mutation of Tyr⁶⁴⁴ to Ala (Tyr⁶⁴⁹ in human) completely eliminates β2 binding, whereas a Tyr⁶⁴⁴ → Phe mutation, which occurs naturally in PIPKγ-687, is tolerated, although it reduces the affinity of PIPKIγ for β2. This observation can be explained by our structural data, which show that although Tyr⁶⁴⁴ interacts directly (via hydrogen bonding) with Lys-808, it also exhibits a considerable buried surface area of 52.6 Ų. This buried surface area and the associated van der Waals interactions are not affected by the Tyr⁶⁴⁴ → Phe mutation.

We also observed at least three major differences between the previously characterized association of an Eps15-derived peptide fragment with the β2 appendage (23) and our structure reported here for PIPKIγ-p90. First, in contrast to Eps15, which exhibits a turn around its glycine residue, the p90-derived peptide wraps around the sandwich subdomain much further and thus covers a considerably larger interface than the short Eps15 sequence (773.7 versus 395.2 Ų; Table 7). Second, because of the substitution of Phe⁶ for glutamate within the Eps15 peptide of p90, the hydrophobic contact with Val-813 is lost. Third, Asp⁸ within Eps15, the side chain of which contacts Tyr-815 of β2, is exchanged for serine in p90 (Fig. 1C and Tables 2 and 3).

TABLE 7.

Interface statistics

The protein interfaces, surfaces, and assemblies (PISA) service at European Bioinformatics Institute (Krissinel and Henrick (43)) was used. iNat and iNres indicate the number of interfacing atoms and residues, respectively, in the corresponding structures.

Peptide	iNat	iNres	Protein	iNat	iNres	Interface area
						Å2
YFPTDERSWVYSPLH	71	14	AP-2(β)	98	25	773.7
SFGDGFADF	35	7	AP-2(β)	55	17	395.2
SWVYSPLH	54	8	AP-2(μ)	75	22	575.4
YECL	50	9	AP-2(μ)	72	21	527.5

In agreement with the critical role of β2 Tyr-815 and Lys-808 in complex formation with the p90 peptide, we observed a complete loss in the ability of GST-tagged β2 Y815A or K808A mutants to pull down PIPKIγ-p90 from rat brain extracts (Fig. 2 A). Conversely, mutation of either Phe⁶⁴⁰ or Trp⁶⁴⁷ to alanines completely eliminates the interaction between GST-PIPKIγ-p90-(451–668) with purified His₆-β2 (Fig. 2B). Somewhat surprisingly, a Trp⁶⁴⁷ → Phe substitution also reduces complex formation below the detection limit in these experiments. A possible explanation is the ability of the indol nitrogen of Trp⁶⁴⁷ to function as a hydrogen donor to Gln-756. Although not detectable on the basis of current electron density, there is the possibility of a positional disorder of the amine and oxyl groups of Gln-756 resulting in formation of a hydrogen bond (distance = 3.38 Å) between Trp⁶⁴⁷ and Gln-756. Binding of GST-PIPKIγ-p90 to talin, another known ligand of the same peptide sequence (9, 10), is unaffected by the Phe⁶⁴⁰ → Ala mutation but is eliminated by a Trp⁶⁴⁷ → Ala substitution, in accordance with previous experiments. In summary, these data establish a firm structural basis for the interaction between PIPKIγ-p90 and the β2 appendage domain of AP-2 and identify Phe⁶⁴⁰ → Ala as a β2-adaptin interaction-defective mutant of PIPKIγ-p90.

FIGURE 2.

Mutants of β2 appendage and PIPKIγ-p90-(451–668) confirm the structural data. A, affinity chromatography of native endogenous PIPKIγ from rat brain synaptosomal extracts using the GST-β2-ear (appendage) domain. Aliquots of rat brain synaptosomal extract and affinity-purified material were analyzed by immunoblotting with antibodies against PIPKIγ or actin as a control. std., 7.5% of the total amount of rat brain synaptosomal extract added to the assay as the standard. B, GST-PIPKIγ-p90-(451–668) WT or mutant proteins were incubated with purified His₆-talin FERM domain or His₆-β2 appendage. The bound talin FERM domain or β2 appendage was detected by immunoblotting using His₆ tag-specific antibodies. std., 50% (talin-FERM) or 10% (β2 appendage) of the total amount of His₆ tagged-protein added to the assay as the standard.

Structural Basis for the Association of PIPKIγ-p90 with AP-2μ

In addition to the association of the PIPKIγ-p90 tail with the β2 appendage of AP-2, it has also been reported that PIPKIγ-p90 binds to the medium chain (μ) of both the AP-1B (30) and AP-2 complexes (22) via a YXXØ-based motif contained within the 28-amino acid splice insert of p90 that overlaps with the β2 appendage binding site. We synthesized the corresponding peptide (SWVYSPLH, with key residues implicated in complex formation being highlighted) and used it for co-crystallization experiments with purified μ2-(158–435) (C-μ2). Crystals were obtained under conditions similar to those seen previously for other YXXØ motif-containing peptides complexed with C-μ2, and these diffracted to a maximal resolution of 2.60 Å (Table 4). Electron density was observed for the entire peptide sequence. As expected, the p90 peptide was found associated with the known YXXØ motif binding site within subdomain A of C-μ2 (Fig. 3A). We observed a three-pin-plug interaction involving Trp⁶⁴⁷, Tyr⁶⁴⁹, and Leu⁶⁵² with Asp-176, Trp-421, and Arg-423 of C-μ2. Trp⁶⁴⁷ is seen in a hydrophobic pocket surrounded by Glu-391, Gln-318, and Pro-393 (Fig. 3B). These interactions very much resemble the complex between a peptide derived from the cytoplasmic loop of the GABA receptor γ2 subunit and C-μ2 (25), where a tyrosine (Tyr⁴) is seen to accommodate the hydrophobic pocket occupied by Trp⁶⁴⁷ in the case of the p90 peptide (Fig. 3C). Hence, both of these complexes also share a similarly large interaction area between peptide and protein ligand (Table 7). Thus, the PIPKIγ-p90 tail domain interacts with AP-2μ via a conventional YXXØ-based sorting motif normally found in transmembrane cargo proteins.

Interaction between the PIPKIγ-p90 Tail and β2 Appendage or C-μ2

Our structural data clearly indicate that binding of the PIPKIγ-p90 tail to the β2 appendage and to C-μ2 is mutually exclusive. We thus next asked which of these two interactions might be favored thermodynamically in vitro. To this aim we performed ITC experiments using a synthetic p90 tail peptide or a β2 binding-defective mutant thereof (Fig. 4A). We determined K_D values of 6 μm for μ2 (Fig. 4, C and D) and 22 μm for β2 appendage domain-containing (Fig. 4, B and D) complexes. As expected the mutation of Phe⁶⁴⁰ to Ala reduced binding to β2 to nearly undetectable levels, whereas association with μ2 was almost unaffected (data not shown). Thus, at least in vitro, the interaction between PIPKIγ-p90 tail and μ2 is thermodynamically favored over that with the β2 appendage. This does not rule out the possibility that complex formation between PIPKIγ-p90 tail and the β2 appendage might be physiologically relevant, nor do these data exclude further regulatory mechanisms, i.e. phosphorylation events that could alter the availability or affinity of these binding sites under physiological conditions (2). For example, interaction of the PIPKIγ-p90 tail with AP-2μ might require its prior phosphorylation by the clathrin-coated vesicle-associated protein kinases AAK1 or GAK in order to induce a conformational opening of the AP-2 complex (2, 3, 16).

FIGURE 4.

Binding of PIPKIγ-p90-derived peptides to the AP-2 subunitsμ2 and β2-ear. ITC of AP-2 subunits μ2 and β2-ear interacting with PIPKIγ-p90-derived peptide. A, diagram showing the domain structure of human PIPKIγ-p90 and the sequence of the PIPKIγ-p90-derived peptide. B and C, differential heating power (Δp) versus time (t) obtained by injecting PIPKIγ-p90-derived peptide into β2-ear (B) or μ2 (C). Initial 5-μl injections were excluded from the fitting procedure. All ITC data were recorded at 25 °C. D, integrated, normalized, and dilution-corrected heats of reaction (Q) versus molar peptide/protein ratio (R). Heats of reaction obtained by injecting peptide into β2-ear (squares) or μ2 (circles) were fitted by a one-site binding model (solid lines) yielding the K_D values shown.

PIPKIγ-p90 Associates with Native AP-2 via Multiple Interaction Determinants

To determine whether sequences contained within the 28-amino acid splice insert of PIPKIγ-p90 represent the only AP-2 binding sites with the PIPKIγ tail domain, we analyzed the ability of GST-PIPKIγ-(451–668) mutant fusion proteins to affinity-purify native AP-2 from rat brain synaptosomal protein extracts. Surprisingly, alanine substitution of Phe-640 (eliminating association with the β2 ear) together with Tyr-649 (a key residue involved in association with AP-2μ) or Trp-647 in conjunction with Tyr-649 or Tyr-649 and Leu-652 significantly reduced but did not completely abrogate AP-2 binding (Fig. 5A). As expected from the crystal structure and the analysis of single point mutants (see Fig. 2), these mutations eliminated association with the β2 appendage (Fig. 5B). All mutants, however, retained the ability to bind to His₆-C-μ2 (Fig. 5C), suggesting that the residual AP-2 binding activity seen in affinity chromatography experiments is because of the association of PIPKIγ-(451–668) with the μ2 subunit. GST-PIPKIγ-p90-(451–668) F640A/Y649A also bound to talin, whereas the W647A/Y649A or the W647A/Y649A/L652A mutant did not (Fig. 5D). These results therefore suggest the existence of a second AP-2μ binding site within the PIPKIγ-p90 tail.

FIGURE 5.

The binding sites for talin and the AP-2 subunits μ2 and β2-ear overlap within the C-terminal splice insert of PIPKIγ-p90. A, affinity purification of native endogenous AP-2 from rat brain extract using PIPKIγ-p90-(451–668) WT or the indicated mutants as bait. Aliquots of rat brain extract and affinity-purified material were analyzed by immunoblotting with antibodies against AP-2 and actin as a control. std., 5% of the total amount of rat brain extract added to the assay as the standard. B, GST-PIPKIγ-p90-(451–668) WT or the indicated mutants were incubated with purified His₆-β2-ear (appendage). Bound β2 appendage was detected immunoblotting using a His₆ tag-specific antibody. std., 10% of the total amount of His₆ fusion protein added to the assay. Bottom, Ponceau S-stained membranes. Molecular mass standards are indicated on the left. Note that the His₆-β2 appendage is not visible in the Ponceau S-stained material due to the presence of GST-PIPKIγ-p90-(451–668) degradation products. C, GST-PIPKIγ-p90-(451–668) WT or the indicated mutants were incubated with purified His₆-C-μ2. Bound C-μ2 was detected by immunoblotting using a His₆ tag-specific antibody. std., 50% of the total amount of His₆-μ2 domain added to the assay as the standard. Bottom, Ponceau S-stained membranes. Molecular mass standards are indicated on the left. D, GST-PIPKIγ-p90-(451–668) WT or the indicated mutants were incubated with purified His₆-talin FERM domain. Bound talin FERM domain was detected by immunoblotting using a His₆ tag-specific antibody. std., 50% of the total amount of His₆-Talin FERM domain added to the assay as the standard. Bottom, Ponceau S-stained membranes. Molecular mass standards are indicated on the left.

Inspection of the primary sequence of PIPKIγ-p90-(451–668) revealed the presence of a second putative YXXØ-based sorting motif between amino acids 497 and 500 (Fig. 6A). To analyze the importance of this proximal YXXØ motif for complex formation between the PIPKIγ-p90 tail domain and AP-2, we mutated the key conserved residues Tyr-497 and Leu-500 to alanines. Whereas GST-PIPKIγ-p90-(451–668) Y497A/L500A bound to AP-2μ, the β2 appendage, or talin with an efficiency similar to the wild-type protein, a mutant in which both YXXØ-based AP-2 binding motifs along with Trp-647 had been mutated to alanines (Y497A/L500A/W647A/Y649A/L652A) did not associate with any one of these ligands (Fig. 6B). To assess the relative contribution of the binding sites for AP-2β and AP-2μ within the PIPKIγ-p90 tail domain, we next performed affinity chromatography experiments from rat brain extracts. Mutational inactivation of the binding site for the β2 appendage domain (F640A) alone or in combination with the distal YXXØ motif within the 28-amino acid splice insert of the tail domain (W647A/Y649A/L652A) somewhat reduced but did not abolish association of GST-PIPKIγ-p90-(451–668) with native AP-2 complexes. Mutational inactivation of the proximal YXXØ motif (Y497A/L500A) did not affect the efficiency with which either AP-2 or talin was pulled down from brain extracts. However, a mutant in which both YXXØ-based motifs along with the β2 appendage binding site had been inactivated (Y497A/L500A/W647A/Y649A/L652A) did not associate with native AP-2 above background levels (Fig. 6C), corroborating our analysis using purified proteins. Hence, the PIPKIγ-p90 tail domain associates with AP-2 via two distinct AP-2μ-binding tyrosine-based sorting motifs, the distal of which overlaps with the binding site for the AP-2β appendage.

FIGURE 6.

PIPKIγ-p90 contains a second tyrosine-based motif within its tail. A, diagram depicting the domain structure of human PIPKIγ-p90. Note the presence of a second YXXØ-based sorting motif within the proximal region of the tail domain. B, affinity chromatography experiments using GST-PIPKIγ-p90-(451–668) WT or mutants were done as described in the legend for Fig. 5. Bound His₆-β2 appendage, -C-μ2, or -talin FERM domain was analyzed by immunoblotting using anti His₆ antibodies. std., 50% (talin-FERM or C-μ2) or 10% (β2-ear) of the total amount of protein added to the assay as the standard. C, affinity purification of native endogenous AP-2 from rat brain synaptosomal extracts using PIPKIγ-p90-(451–668) WT or the indicated mutants as bait. Aliquots of rat brain synaptosomal extract and affinity-purified material were analyzed by immunoblotting with antibodies against AP-2, talin, or actin as a control. std., 10% of the total amount of rat brain synaptosomal extract added to the assay as the standard. Splice is indicated by solid line (all taken from the same exposure). D, affinity purification of full-length HA-tagged PIPKIγ-p90 variants (WT or mutants) from transiently transfected COS-7 cells using GST, GST-C-μ2, or GST-β2-ear as a matrix. Aliquots of the total cell extracts and of the affinity-purified material were analyzed by immunoblotting with antibodies against actin or HA. Std., 12% of the total amount of cell extract added to the assay as the standard. Bottom, Ponceau S-stained membranes. Molecular mass standards are indicated on the left and GST fusion proteins on the right. E, quantification of the amount of HA-PIPKIγ-p90 variants affinity-purified on GST-C-μ2 (left) or GST-β2-ear (right). The total fraction of PIPKIγ-p90 (as % of input) found in the affinity-purified material as exemplified in D was quantified using ImageJ. Data are given as mean ± S.E. (n = 2 independent experiments).

Finally, we wanted to assess the contribution of these various determinants within the kinase tail to the association of native full-length PIPKIγ-p90 with AP- 2. To this aim, we performed affinity chromatography experiments from transfected COS-7 cells using GST-C-μ2 or GST-β2 appendage domain as baits. As expected, mutational inactivation of the β2- ear binding site within PIPKIγ-p90 (F640A; Δβ2 site) completely eliminated association with the β2 appendage, whereas binding to AP-2μ was unaffected (Fig. 6D and E, Δβ2 site). A PIPKIγ-p90 mutant in which both YXXØ-based motifs along with the β2 appendage binding site within the tail had been inactivated (Y497A/L500A/W647A/ Y649A/L652A; Δβ2μ2 sites) did not bind to the GST-β2 appendage but retained the ability to form a complex with AP-2μ, although with somewhat reduced efficiency (Fig. 6, D and E, Δβ2μ2 sites). These results indicate that complex formation between AP-2 and PIPKIγ-p90 is, at least in part, driven by determinants outside of the PIPK tail domain, presumably by the association of its catalytic core with AP-2μ (17). This conclusion also agrees with the dissociation constants measured for these various sites (22 μm for PIPKIγ-p90 tail-β2 ear; 6 μm for PIPKIγ-p90 tail-C-μ2 (compare Fig. 4); 0.5 μm for PIPKIγ core-C-μ2 (17)). Our data therefore argue in favor of a model according to which multiple binding sites contribute to complex formation between PIPKIγ-p90 and the AP-2 complex.

A YXXØ Motif-containing Peptide Derived from the p90 Tail Can Stimulate the PI(4,5)P₂-synthesizing Activity of AP-2μ-bound PIPKIγ

Multiple interactions of AP-2 with PIPKIγ-p90, in addition to recruiting the kinase to sites of endocytosis, may serve to regulate enzymatic activity. In fact, it has been reported that receptor-derived YXXØ motif-containing peptides potently stimulate the PI(4,5)P₂-synthesizing activity of PIPKIγ bound to AP-2 (17). The presence of YXXØ motifs within the PIPKIγ-p90 tail domain could thus serve as an intramolecular switch to regulate PI(4,5)P₂ formation by PIPKIγ-p90·AP-2 complexes. To test this possibility we prepared lysates from stably transfected HEK293 flip-in cells expressing HA-PIPKIγ-p87 (i.e. a variant of PIPKIγ lacking the AP-2μ/β-binding splice insert) and analyzed lipid kinase activity. PI(4,5)P₂ synthesis in untreated lysates was comparably weak, and this did not change upon adding His₆-C-μ2 (Fig. 7A). The addition of the PIPKIγ-p90-derived YXXØ motif-containing tail peptide together with His₆-C-μ2 stimulated the formation of radiolabeled PI(4,5)P₂, similar to that seen with an AP-2μ-binding sorting signal peptide (FYRALM, with key residues being highlighted) derived from the epidermal growth factor (EGF) receptor. A mutated nonfunctional peptide lacking the ability to bind to C-μ2 was inactive (Fig. 7B). Peptides were without effect in the absence of C-μ2 (data not shown; see Ref. 17). Similar results were obtained when the kinase activity of HA-PIPKIγ-p87 was determined following affinity purification on a GST-C-μ2-based matrix in the presence or absence of peptides (supplemental Fig. 1). These results are consistent with a model according to which an AP-2·PIPKIγ-p90 complex provides a local pool of PI(4,5)P₂ at endocytic sites, i.e. during SV recycling.

FIGURE 7.

PIPKIγ-mediated PI(4,5)P₂ synthesis is stimulated by a tyrosine motif peptide derived from the p90 tail domain. A, detergent lysates of HEK293 flip-in cells stably expressing HA-PIPKIγ-p87 were incubated in the presence or absence of 2 μm His₆-C-μ2 and the indicated peptides (100 μm), phosphatidylinositol 4-phosphate and γ-[³²P]ATP. Extracted lipids were separated by TLC, and the amount of [³²P]PI(4,5)P₂ generated was detected by phosphorimaging analysis. Representative data are shown. p90 insert, AP-2μ-binding YXXØ motif-containing peptide derived from the splice insert of PIPKIγ-p90 (YFPTDERSWVYSPLH, CT-peptide); FYRALM, AP-2μ-binding YXXØ motif-containing peptide derived from the epidermal growth factor receptor (YXXØ peptide); FARALM, AP-2μ-binding defective mutant version thereof (AXXØ peptide). B, data derived from two independent experiments as shown in A were analyzed to quantify PIPKIγ-mediated [³²P]PI(4,5)P₂ synthesis in the absence or presence of the indicated peptides (mean stimulation ± S.E.). Values were normalized to the amount of PI(4,5)P₂ synthesized in the absence of peptide.

Inhibition of SV Endocytosis by Expression of the PIPKIγ-p90 Tail Domain Depends on Its Ability to Associate with AP-2

PIPKIγ plays an important role in the exo-/endocytic cycling of SVs in primary neurons (20). Loss of PIPKIγ expression in mice severely impairs clathrin-mediated endocytosis of SVs, resulting in impaired retrieval of SV proteins from the presynaptic plasmalemma. A similar phenotype is observed upon expression of the PIPKIγ-p90 tail domain in hippocampal neurons in culture (21). These latter findings enabled us to functionally analyze the contribution of the various AP-2 binding sites within the PIPKIγ-p90 tail domain to its ability to impair SV endocytosis in primary neurons. To this aim we made use of synapto-pHluorin, a widely used tracer for SV exo-/endocytosis. Fluorescence of synapto-pHluorin is critically dependent on the pH and can thus be used to quantitatively analyze partitioning of the protein between the plasmalemma and intracellular SV-localized pools. Fluorescence changes were monitored from active synaptic boutons (i.e. displaying appropriate responses to electrical stimulation; compare Fig. 8A) expressing synapto-pHluorin together with mRFP (as a control) or mRFP-tagged variants of the PIPKIγ-p90 tail. Fluorescence quantification after acid quenching and ammonium dequenching then revealed the relative ratio of vesicular-to-surface-stranded pHluorin molecules (Fig. 8B). An increased fraction of surface-stranded synapto-pHluorin is indicative of impaired SV endocytosis at active boutons. In agreement with previous results using FM4-64 to monitor SV endocytosis (21), we observed that overexpression of a fusion protein comprising the C-terminal tail of PIPKIγ-p90 tagged with mRFP significantly increased the surface-stranded pool of synapto-pHluorin (Fig. 8C) by more than 50%. By contrast, overexpression of the same construct had no significant effect on transferrin internalization (supplemental Fig. 2), in agreement with the specific role of PIPKIγ-p90 in the regulation of SV exo-/endocytosis. Mutational inactivation (F640A) of the interaction site for the β2 appendage domain within the PIPKIγ-p90 tail only modestly affected its ability to impair SV endocytosis (Fig. 8C, Δβ2 site). A PIPKIγ-p90 tail mutant unable to interact either with the β2 appendage or with AP-2μ had nearly completely lost its dominant-negative effect on synapto-pHluorin retrieval from the cell surface (Fig. 8C, Δβ2Δμ2 sites). Whether the remaining small degree of inhibition of SV endocytosis seen upon expression of this mutant construct resulted from sequestration of other yet unidentified binding partners of the PIPKIγ tail or reflected nonspecific effects remains to be determined. In any case, these data corroborate our structural and biochemical analysis and further support an important physiological role for complex formation between AP-2 and PIPKIγ in SV endocytosis, a function that is, at least in part, regulated by AP-2-binding determinants within the kinase tail domain.

FIGURE 8.

The ability of the PIPKIγ tail to interfere with SV endocytosis in primary hippocampal neurons is dependent on its interaction with AP-2β and AP-2μ. A, synapto-pHluorin fluorescence before (top left, pH 7.4) and immediately after (top right) stimulation with 200 action potentials (AP) to prove viability. Challenge with acidic solution (pH 5.5) results in fluorescence quenching (bottom left), whereas neutralization with NH₄Cl produces maximum fluorescence (bottom right). Scale bar, 10 μm. Fluorescence values are color-coded as depicted on the scale. B, average synapto-pHluorin trace for primary hippocampal neurons expressing mRFP. The illustration shows the fluorescence of synapto-pHluorin during various steps in the experimental protocol. Fluorescence in >50 synaptic boutons was normalized to the fluorescence intensity observed under physiological pH ± S.E. C, on the basis of the relative fluorescence values measured before and after acid quenching/dequenching, the vesicular-to-surface-stranded pool ratios of synapto-pHluorin were calculated. Data for neurons expressing different mRFP-PIPKIγ-p90 tail domain constructs were normalized to the mRFP control and plotted ± S.E. (n = 4 experiments). Expression of mRFP-tagged PIPKIγ-p90 tail WT tail results in a significant increase in the surface pool compared with control neurons expressing mRFP. This effect is gradually reduced in mutants lacking the ability to associate with the AP-2 β-ear (Δβ2 site) or both AP-2μ and β2-ear domains (Δβ2μ2 sites). Control, 670 synaptic boutons, 13 neurons; WT (wt), 621 synaptic boutons, 10 neurons; Δβ2 site, 463 synaptic boutons, 8 neurons; Δβ2μ2 sites, 548 synaptic boutons, 8 neurons). Error bars indicate S.E. (control, ±0.0177; WT, ±0.0237; Δβ2 site, ±0.0255; Δβ2μ2 sites, ±0.0215; *, p < 0.01; **, p < 0.001).

DISCUSSION

In the present study we have provided a firm structural basis for the association of the PIPKIγ-p90 tail domain with AP-2μ and the β2 appendage domain, respectively. We show that these interactions with different subunits and domains of AP-2 partly involve overlapping determinants within the 28-amino acid splice insert of the PIPKIγ-p90 tail. Both complexes largely depend on hydrophobic/aromatic amino acid-in-groove interactions. Specifically, the PIPKIγ-p90 tail peptide is seen to wrap around the side site of the sandwich subdomain of the β2 appendage in a position similar but not identical to that occupied by a peptide derived from the accessory protein Eps15 (23). As this site is a minor protein binding interface within AP-2, this mechanism might allow for privileged access of PIPKIγ-p90 to the endocytic machinery. The same peptide can also accommodate the sorting signal binding domain of AP-2μ via a three-pin-plug interaction involving Tyr-649, Leu-652, and Trp-647, the latter a residue known to be required for the association of PIPKIγ-p90 with the actin-associated scaffolding protein talin at sites of cell adhesion or synaptic contacts (9, 10). The use of partly overlapping binding sites for AP-2μ, the β2 appendage, and talin suggests that these various interactions may be subject to regulation during exo-/endocytic cycling of SVs. In agreement with this proposal, PIPKIγ-p90 has been shown to undergo activity-dependent Cdk5-mediated phosphorylation at Ser-650 (corresponding to Ser-645 of mouse PIPKIγ-p90) during SV exo-/endocytosis (33), a modification that has been shown to impair its association with talin (33) and with the β2 appendage domain of AP-2 (21). Our crystallographic data (Table 2) reveal the molecular basis for this regulation. Ser-650-OH participates in hydrogen bonding with the peptide backbone of Phe-753 and Leu-770, interactions that are likely to be compromised by phosphorylation of Ser-650. Phosphorylation of Tyr-649 by Src or other tyrosine kinases has been shown to facilitate association of PIPKIγ-p90 with talin (34). Our structural data presented here would argue that the same modification should inhibit binding of the PIPKIγ-p90 tail to AP-2μ or the β2 appendage, suggesting the existence of a phosphorylation-dependent cycling of PIPKIγ-p90 between sites of cell adhesion and endocytosis. Mutagenesis paired with biochemical binding experiments also suggests that complex formation between AP-2 and PIPKIγ-p90 is dependent in part on determinants outside of the p90 tail domain, presumably involving the direct association of AP-2μ with the central kinase core (17).

The clathrin-based endocytic machinery together with the p90 splice isoform of PIPKIγ is concentrated within the presynaptic compartment where SVs undergo activity-dependent rapid cycles of exo-/endocytosis (4, 32). It is therefore tempting to speculate that the presence of additional binding sites within the 28-amino acid splice insert of PIPKIγ-p90 for AP-2β/μ serves to concentrate the enzyme at endocytic hot spots near the active zone. According to a hypothetical model (Fig. 9), one might envision that the initial recruitment of PIPKIγ-p90 to sites of SV endocytosis involves binding of the YFPTDERSWVYSPLH (with key residues being highlighted) sequence within the p90 tail to the AP-2 β appendage (Fig. 9A), which acts as an autonomous platform for the enrichment of endocytic accessory factors (35). Complex formation might be aided by the association of the PIPKIγ core with AP-2μ and/or by phosphorylation of AP-2μ. Phosphorylation of μ2 at Thr-156 facilitates a conformational opening of the AP-2 complex and the dislocation of the C-terminal sorting signal binding domain of μ2 from the remainder of the AP-2 core (16). This conformational change would enable C-μ2 to bind to YXXØ-based sorting signals of transmembrane cargo proteins (Fig. 9B) or to the SWVYSPL and RSYPTLED peptide motifs within the PIPKIγ-p90 tail (Fig. 9C). Occupation of the YXXØ motif binding site on AP-2μ likely results in a potent stimulation of PIPKIγ-mediated PI(4,5)P₂ synthesis (compare Fig. 7). Recent in vitro experiments suggest that clathrin assembly might facilitate displacement of the PIPKIγ-p90 tail from the β2 appendage, thereby potentially providing directionality to the process of complex assembly and disassembly (36). Clearly, further experiments are needed to put such a hypothetical scenario to the test.

FIGURE 9.

Hypothetical model illustrating molecular determinants and possible mechanisms of association regulating complex formation between AP-2 and PIPKIγ-p90. A, PIPKIγ-p90 may initially be recruited to or stabilized at endocytic sites by association of its tail with the β2 appendage domain of AP-2. Binding is comparably weak (K_D = 22 μm) and will require stabilization or modulation by further contacts. B, for example, YXXØ motif-containing cargo recognized by AP-2μ at PI(4,5)P₂-containing plasmalemmal hot spots might aid the formation of a heteromeric complex involving binding of the PIPKI core to AP-2μ, an association that facilitates the PI(4,5)P₂-synthesizing activity of PIPKIγ-p90. C, PIPKIγ-p90 may also contribute to AP-2-dependent sorting of SV cargo lacking conventional YXXØ motifs at presynaptic sites. This might involve the association of YXXØ-based motifs within the PIPKIγ-p90 tail domain with AP-2μ, thereby facilitating local PI(4,5)P₂ production.

One unresolved question concerning clathrin-mediated SV endocytosis pertains to the mechanism of SV cargo recognition by adaptor proteins including AP-2, endophilin, and stonin 2 (37). It has long been known that SV proteins and conventional cargo such as the transferrin receptor segregate during their endocytic itinerary in neurons or neuroendocrine cells (38). Moreover, accumulating evidence suggests that SV proteins are sorted into clathrin-coated vesicles by cargo-specific, partly YXXØ motif-independent mechanisms (39). These include the recognition of VGLUT1 by the endocytic SH3 domain protein, endophilin (40), or the internalization of synaptotagmin 1 by its dedicated sorting adaptor, stonin 2, via sorting determinants contained in its C2 domains (41). Association of AP-2μ with YXXØ peptide motifs of presynaptically enriched PIPKIγ-p90 might thus provide a potential means to 1) exclude conventional YXXØ motif-containing cargo such as growth factor or nutrient receptors from presynaptic clathrin-coated pits formed during SV retrieval and 2) activate PIPKIγ-p90 to generate a pool of PI(4,5)P₂ that allows for efficient AP-2/clathrin coat formation during SV endocytosis. Further studies capitalizing on the molecular insights provided here will need to address this possibility.