Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus–containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2–17 of ARF1 ([2–17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2–17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

their PH domains binding to membrane components, including phosphoinositides and GTPases (20,24,26,27,30,(33)(34)(35). Recruitment concentrates and orients the protein on a surface that contains a target molecule. In addition, PH domains can autoinhibit or position other structural elements of a protein to inhibit intramolecular catalytic domains, as described for kinases and guanine nucleotide exchange factors (36 -41). For p63RhoGEF, G␣ q binding to a C-terminal extension of the PH domain relieves autoinhibition (36). In the case of the Arf exchange factors, cytohesins, cooperative binding of phosphoinositide and Arf6⅐GTP or Arl4⅐GTP to the PH domain relieves PH domain-mediated autoinhibition (29,39,42). Finally, as is observed in some exchange factors, PH domains or extensions of the PH domain can contribute directly to binding the substrate protein (43,44).
The PH domain of ASAP1 might contribute to GAP activity by the third mechanism, through a direct interaction with the substrate. We have found recruitment of the Arf GAP domain to a hydrophobic surface containing the substrate Arf1⅐GTP is not sufficient for GAP activity, and the cognate PH domain is necessary (16). We have also found that GAP activity requires a unique structural feature of Arf family GTPase, the N-terminal extension from the GTP-binding domain, which has previously been found to bind to ASAP1 (although the binding site has not been determined) (45). Furthermore, the interaction might be regulated by PIP 2 . PIP 2 binding to the PH domain is necessary for activity (14,15,17,46). These observations, together with the precedent of cytohesin, in which a phosphoinositide, phosphatidylinositol 3,4,5-trisphosphate, regulates binding of Arf6⅐GTP to the PH domain (29), have led us to hypothesize that, rather than mediating recruitment to a lipid bilayer, PIP 2 binding to the PH domain of ASAP1 regulates binding to the N terminus of Arf1 to control GAP activity.

PIP 2 -dependent activity is independent of a lipid surface
The myristoylated N terminus of Arf1 mediates recruitment to surfaces (49 -51) and binds to PIP 2 (52,53). Therefore, part of the difference in GAP activity against full-length myristoylated Arf1 and [⌬17]Arf1 might be due to lack of recruitment of [⌬17]Arf1 to the surface containing ASAP1. To determine whether there was a recruitment-independent component of PIP 2 activation, we sought conditions that would allow us to measure activity without LUVs. When bound to GTP, fulllength Arf1 is not stable without a hydrophobic surface, but we have previously identified a point mutant of Arf1, [L8K]Arf1, that is stable without a hydrophobic surface (45).
[L8K]Arf1 is as efficient a substrate as WT Arf1 for ASAP1 when the PIP 2 in the reaction is presented in mixed micelles of Triton X-100 (45,54). [L8K]Arf1 is ϳ10-fold less efficient as a substrate than WT Arf1 when the PIP 2 in the reaction is incorporated into LUVs. However, GAP activity on [L8K] Arf1, like that on WT Arf1, depends on PIP 2 ( Fig. 2A and Table 1). Importantly, the GAP activity of ASAP1 with [L8K]Arf1 as a substrate is inhibited by the [2-17]Arf1 peptide, with an IC 50 of 0.8 Ϯ 0.1 M, but not by the [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]scrambled peptide (Fig. 2B). A second consideration for examining activity without a hydrophobic surface is the soluble PIP 2 analog. In crystal structures of the ligand-bound and unliganded forms of the PH domain (14), PIP 2 binds to two sites, the canonical site and the atypical site. These two binding sites are separated from one another by the loop between ␤-strands 1 and 2, the structure of which is stabilized by isoleucine 353. We considered that there may be a minimal length of the acyl group of PIP 2 required to stabilize the loop and support GAP activity. We tested [L8K]Arf1 as a substrate with either no PIP 2 or 200 M diC4PIP 2 , diC6PIP 2 , or diC8PIP 2 , which all have critical micelle concentrations greater than 1 mM (55,56). 7 We observed that PIP 2 analogs increased activity dependent on acyl length, with more than a 7,000-fold increase in activity with diC8PIP 2 compared with no PIP 2 ( Fig. 2C and Table 2). Titra-tion of diC8PIP 2 revealed a sigmoidal dependence for PIP 2stimulated activity with a Hill coefficient of 1.9 Ϯ 0.27 (S.E.) (Fig. 2D) binding sites on the ASAP1 PH domain (14) and at least one on Arf1 (52,53,58) that are necessary to form the active complex. 8 There were some differences in the specificity of headgroups for stimulating GAP activity between the water-soluble phospholipid analogs and LUVs. For instance, diC8-phosphatidylserine (PS) increased activity by 1000-fold (Table 2), compared with 15-fold for PS in LUVs (14). 9 Nevertheless, the N terminus of Arf1 was critical for the observed activation as it was in LUVs. diC8PIP 2 stimulated the activity of [L8K]Arf1, which has the N-terminal extension from the GTP-binding domain, more than 7,000-fold, but only 3-fold for [⌬17]Arf1, which does not have the extension ( Fig. 2E and Table 3). The cognate PH domain was required as protein composed of the Arf GAP and ankyrin repeat domains (ZA) or the PH domain of PLC␦1 and the Arf GAP and ankyrin repeats of ASAP1 (PdZA, see Fig. 1A for a schematic of the proteins) had little or no activity in the presence of LUVs or diC8PIP 2 (Fig. 2, F and G). As for activity measured with PIP 2 in LUVs, [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]Arf1, but not [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]scrambled, inhibited diC8PIP 2 -stimulated activity with an IC 50 of 4.7 Ϯ 0.4 M (Fig. 2H). Although there are differences between the reaction with diC8PIP 2 and LUVs, which we are pursuing in other studies, 9 our results indicate that the N termini of Arf1 and PH domain are critical for catalysis whether PIP 2 is presented in LUVs or as the soluble analog diC8PIP 2 .

PIP 2 -dependent binding of N terminus of Arf1 to the PH domain of ASAP1 correlates with PIP 2 -dependent GAP activity
As an initial effort to identify the binding determinants between PH ASAP1 and [2-17]Arf1, [2-17]Arf1-TAMRA was incubated with the PH domain of ASAP1 ([325-451]ASAP1), diC8PIP 2 , and the cross-linker EDC. A cross-linked product was identified by SDS-PAGE (data not shown). MS analysis of the product revealed cross-linking between Glu-337 of ASAP1 and Lys-10 of Arf1. The crystal structure revealed that the ASAP1 PH domain has a 17-amino acid N-terminal extension, containing Glu-337, prior to the first ␤-strand in the canonical fold ( Fig. 4B) (14). These residues are part of the linker between the BAR and PH domains. Interdomain linkers and extensions of the PH domain contribute to PH domain binding to proteins, for example in cytohesins (29) and p63RhoGAP (36), respectively. We tested the hypothesis that the N-terminal extension of the PH domain of ASAP1 functions to facilitate binding to Arf1 by investigating the binding of the PH domain, including the 17 residues, without 9 of the 17 residues or without 14 (Fig. 7E). However, there was a 100-fold difference in activity between the proteins, measured with a GAP assay based on the difference in tryptophan fluorescence between Arf1⅐GTP and Arf1⅐GDP ( Fig. 7F) (61). Based on these results, the linker might contribute to binding, but it is not the major determinant. These results are additional support for the idea that simple recruitment of the Arf GAP domain to a surface containing Arf1⅐GTP is not sufficient for efficient activation of GAP activity, as there was less than a 2-fold difference for  We also examined point mutants in an effort to test the prediction that mutations in the PH domain can disrupt [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]Arf1 binding and GAP activity without disrupting PIP 2 binding (Figs. 4B and 8, A-F). We selected residues in the N-terminal extension of the PH domain. Two changes were introduced into the linker: (i) glutamine 331, leucine 332, and glutamine 333 were changed to alanines (Q331A,L332A,Q333A), and (ii) glycine 339 was changed to isoleucine (G339I). In addition, we introduced mutations to change aliphatic residues within the ␣-helix of the PH domain (tyrosine 419 to either alanine or glutamate (Y419A or Y419E), and isoleucine 423 to alanine (I423A). The residues are surfaceexposed and near the N-terminal extension of the PH domain that may contribute to binding the N terminus of Arf1. The 10 High throughput screens for inhibitors using this assay are ongoing.   (Fig. 8G).
[L8K,K10D,K15D,K16D]Arf1 was ϳ1000-fold less efficient a substrate than [L8K]Arf1 (Fig. 8H). The correlation of reduced binding with reduced GAP activity is consistent with our hypothesis, although we cannot exclude that the lack of activity was because the mutant Arf1 did not bind PIP 2 (52).

Mutations in the PH domain that decrease binding of the N terminus of Arf1 also decrease the effect of the full-length ASAP1 on actin remodeling
ASAP1 regulates actin remodeling in cells. One GAP-dependent function is reduction of the formation of circular dorsal ruffles (CDRs) in fibroblasts treated with platelet-derived growth factor (PDGF) (4). If binding of the N terminus of Arf to the PH domain of ASAP1 were important to function, we would predict mutants with reduced binding would be less active in cells. We determined the effect of ectopic expression of WT ASAP1, [R497K]ASAP1 (a mutant with the catalytic arginine changed to lysine, which has less than 1/10,000th the activity of WT ASAP1 (4, 15) (Fig. 9). These results indicate the interaction between the PH domain of ASAP1 and the N terminus of Arf1 is critical for the cellular functions of ASAP1.

Discussion
We examined the mechanism by which PIP 2 binding to the PH domain of ASAP1 modulates activity of the GAP domain. In previous studies, we found that both the cognate PH domain of ASAP1 and the N-terminal 16 amino acids of Arf were necessary for PIP 2 -stimulated GAP activity. Here, we report that (i) one mechanism by which the PH domain regulates GAP activity is independent of recruitment to membranes, (ii) PIP 2 binds to the PH domain to regulate binding to the N terminus of the substrate Arf1⅐GTP, and (iii) disrupting the Arf1 N terminus-PH domain association reduces GAP activity. Mutations that reduced binding of the N terminus of Arf1 to ASAP1 also reduced the activity of ASAP1 in cells. The results are consistent with our hypothesis that PIP 2 regulates binding of the N terminus of Arf1 to the PH domain of ASAP1, which might underlie the high degree of regulation of the catalytic activity.
The PH domain of ASAP1 contributes to enzymatic activity primarily by one of the three distinct mechanisms that have been described for PH domains. The first mechanism we had investigated was recruitment to a membrane containing a target protein (20). Our previous studies had established that recruitment was not sufficient (16,17), and our results examining the effect of diC8PIP 2 and comparing the activity of [325-724]ASAP1 with [339 -724]ASAP1 reported here are additional evidence that recruitment is not the primary mechanism by which ASAP1 is activated by PIP 2 . PH domains have also been found to position autoinhibitory motifs in Grp1 and Akt such that deletion of the PH domain increases activity (37,39). Similarly, the PH domain in SOS1 occludes the DH domain (41), the PH domain of p63RhoGEF autoinhibits the DH domain (36), and two PH domains occlude the active site in the nucleotide exchange factor Farp2 (40). Autoinhibition is relieved by G␣ q binding to a C-terminal extension of the PH domain of p63RhoGEF (36) and Arf6 binding to the PH domain and N-and C-terminal linkers on the PH domain of cytohesin (29,39,42). For ASAP1, deletion of the PH domain results in loss of activity (17), leading us to conclude that autoinhibition by the PH domain is not a feature of the regulation of ASAP1. Our results indicate that the PH domain of ASAP1 contributes by a third mechanism by which PH domains can affect an adjacent enzymatic domain by directly interacting with the substrate of the catalytic domain. PIP 2 binding to two sites within the PH domain of ASAP1 (14) is necessary for activa- shown. Lysine 342, the first residue of the ␤1 strand of the PH domain, is indicated as well as residues that were changed for experiments presented in Fig. 5 (62). For Brag2, an Arf-exchange factor, switch 1 of Arf binds to an expansion of the PH domain, formed by the linker between sec7 and the PH domain (43). ASAP1 is distinct from these in that the PH domain binds to the N-terminal extension of Arf1.
Like the PH domains of cytohesins, the ASAP1 PH domain binds to Arf under the control of PIP 2 (29,39,42), but the mechanism is distinct. Switch 1, the interswitch domain, and switch 2 contribute to binding to the PH domain of the cytohesin Grp1. In contrast, the N terminus of Arf binds to the PH domain of ASAP1, although we cannot exclude some involvement of switch 1 or 2. Another difference is that binding to the cytohesin PH domain is driven by binding a single phosphoinositide molecule. For ASAP1, simultaneous binding of two phospholipids to a single PH domain is necessary. A third difference is that Arf binding to the PH domain of cytohesin 3 (Grp1) involves linkers on both the N-and C-terminal regions of the PH domain. Although there is some contribution of an interdomain linker, the linker is not required for binding between the ASAP1 PH domain and Arf1.
We investigated the role of a 17-amino acid extension of the PH domain based on the precedent of cytohesins (29,39,42) A B Figure 5. [2-17]Arf1-ASAP1 association is specific for PH ASAP1 as measured by fluorescence anisotropy. A, principle of fluorescence anisotropy. A molecule with a linked fluorophore is excited with plane polarized light. If the molecule tumbles rapidly, relative to the lifetime of the excited fluorophore such that the fluorophore randomly changes its orientation before emission, the emission is depolarized and intensity in perpendicular planes is similar. If the molecule tumbles slowly, e.g. as would occur if it binds to a larger molecule, the emission will be partly polarized, with unequal intensities in perpendicular planes, which is a measure of anisotropy. B, fluorescence anisotropy of TAMRA linked to [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]

PIP 2 regulates Arf1 binding to the ASAP1 PH domain
and p63RhoGEF (36). For cytohesins, linkers N-and C-terminal to the PH domain are critical for binding Arf6. For p63RhoGEF, a C-terminal extension of the PH domain mediates binding to G␣ q . We found that the 17-amino acid N-terminal extension of the PH domain had a minimal contribution to binding the N terminus of Arf1 but was nevertheless critical for GAP activity, independent of both PIP 2 binding and peptide binding. This result provides additional support for the notion that the PH domain is not simply a recruitment mechanism as the PH domain without the linker was recruited to LUVs by PIP 2 nearly as well as the PH domain with the linker, but PZA without the linker had a fraction of the activity of PZA containing the linker. In addition, the results indicate that there is a difference in regulation of GAP activity by PIP 2 in a membrane compared with a soluble analog of PIP 2 , with a larger differential in activity with and without the linker observed with LUVs than with diC8PIP 2 . Possible mechanisms by which the N-terminal extension of the PH domain affects the GAP domain and how this is influenced by a lipid bilayer are currently being explored.
Other PH domains bind to protein and phospholipids simultaneously but cooperativity similar to that seen in cytohesin and ASAP1 has not been reported. One example is the phosphotyrosine binding (PTB) domain 11 of disabled-1 (Dab1), which binds simultaneously to PIP 2 and peptides containing Asn-Pro-Xaa-Tyr found in, for example, amyloid precursor protein and the apolipoprotein E receptor 2 (64,65). Dab1 binds to PIP 2 through a site that aligns with the PIP 2 -binding site of the PH domain of PLC ␦1. The peptide-binding site is separate and involves the ␤5-strand and the C-terminal ␣-helix. Other than by restricting the protein to the cellular site with the target peptide, PIP 2 does not influence peptide binding (65). Another example of a PH domain that binds simultaneously to a phosphoinositide and protein is the PH domain of FAPP1 (Four-phosphateadaptor protein 1), which binds to Arf1 and phosphatidylinositol 4-phosphate simultaneously (26,27). Both ligands are on the same surface, which allows coincidence detection; however, we are not aware of evidence for cooperative binding.
The proposed function of the ASAP1 PH domain is similar to models for GTPases (66,67). For both, ligand binding (phosphoinositides or guanine nucleotides) controls the affinity for a target protein. The important difference between PH domains and GTPases is related to the mechanisms for switching between active and inactive conformations. For GTPases, high-affinity binding of nucleotide rapidly activates the protein, whereas rapid inactivation is achieved by a catalytic event, GTP hydrolysis. PH domains do not have a catalytic activity to rapidly convert to the inactive form. The conversion from inactive to active and back is 11 PH, PTB, EVH1, and RanBD have a common fold, referred to as the PH domain superfold (63).

PIP 2 regulates Arf1 binding to the ASAP1 PH domain
controlled by ligand binding and dissociation. Cooperative binding of phospholipids, as reported for ASAP1 (14), may provide rapid allosteric regulation of activity. Our results highlight the possible importance of extensions from the nucleotide-binding domain for signaling in Ras-superfamily proteins. A role of an extension from the nucleotide-binding domain for protein function was described as early as 1991 for Arf family proteins. In Arf, the N-terminal extension from the nucleotide-binding fold (68) is myristoylated and associates with membranes when Arf is bound to GTP. Arf1 without the extension was found to be inactive in vitro and in cell-based functional assays (69,70). A peptide composed of these 16 amino acids of Arf1 blocked in vitro assays of Arf activity (69,71). Several years later, the finding of relative movement of the Arf1 N terminus on GTP binding supported the idea that it functioned as a third switch motif in Arf (50,72). Here, we observe that it binds directly to the GAP, which promotes GAP activity. Efficient interaction of Arf with the exchange factor Brag2 might also involve the N terminus of Arf (43,73). Extensions of other G proteins from the GTPase domain have been found to bind effector proteins. Most relevant for Arf is Arl2 in which the N-terminal helix, switch 1, and switch 2 are all part of the interface with Binder of Arl Two (BART) (74). The C-terminal hypervariable region (HVR), an extension of the nucleotide binding fold, of other Ras superfamily members might be similarly important. In molecular dynamic simulations coupled with FRET measurements in living cells, the accessibility of the HVR of Ras was determined by the bound nucleotide (75)(76)(77). In other studies, Rheb and KRas4b were reported to bind phosphodiesterase through the HVR with no contact with switch 1 or 2 (78,79).

PIP 2 regulates Arf1 binding to the ASAP1 PH domain
In summary, our results support the hypothesis that PIP 2 -dependent binding of the N terminus of Arf1 to the PH domain of ASAP1 regulates GAP activity. In ongoing studies, we will con-tinue to test the hypothesis with full-length ASAP1 in cells and will identify the binding interface and the mechanism by which PIP 2 binding to the PH domain facilitates the interaction.   mol. Chloroform was removed by streaming nitrogen over the solution for 1 h and then placing the tubes in a lyophilizer for an additional hour. Lipids were hydrated with 0.1 to 0.5 ml (volume for final lipid concentration of 5 mM total lipid) of 25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM DTT, with or without 10% sucrose (w/v) as indicated, and then subjected to five freezethaw cycles followed by extrusion through Whatman Nuclepore Track-etched membranes, with 1-m pores, in an Avanti Polar Lipids lipid extruder (82).

GAP assays
GAP activity was determined by measuring the conversion of [␣-32 P]GTP bound to Arf1 to [␣-32 P]GDP as described (80). For experiments in which the GAP was titrated into a reaction, the concentration of GAP required to induce hydrolysis of 50% of the bound GTP in 3 min is referred to as the C 50 . For experiments with a fixed concentration of GAP, activity was expressed as ln(S 0 /S)/t in which S 0 is the initial amount of substrate, Arf⅐GTP, and S is the amount of substrate remaining after a fixed time, t, which was typically 3 min (48).
GAP activity was also determined by following the reduction in tryptophan fluorescence that is observed when Arf⅐GTP is converted to Arf⅐GTP as described (83)(84)(85). In these experiments, [325-724]ASAP1 or [339 -724]ASAP1 was titrated into a reaction containing myrArf1⅐GTP bound to nanodiscs (NDs). The tryptophans were excited at 297 nm, and emission at 340 nm was measured.

FRET
Peptides were labeled with EDANS either at the C or N terminus through an extra glutamic acid. EDANS-labeled peptides and protein were incubated in PBS, pH 7.4, with further additions as indicated in the figure legends and text. The samples were excited at 280 nm and emission spectra recorded from 310 to 540 nm with a Jobin-Yvon Horiba FluroMax-3 spectrofluorimeter. Excitation and emission slit widths were 5 nm. For experiments in which the peptide was titrated, FRET efficiency (E) was calculated from the intensities at 340 nm using Equation 1, where F da is the intensity in presence of acceptor and F d is the intensity without acceptor. In experiments in which the donor (protein) was titrated, emission from the acceptor to shown as a measure of FRET.

Fluorescence anisotropy
Proteins were titrated into a reaction containing 1 M where I ʈ is the parallel emission intensity, I Ќ is the perpendicular emission intensity, and G is the grating factor that corrects for wavelength-dependent distortion of the polarizing system (59,86). Titrations were repeated at least three times. The average anisotropy value for each concentration of protein was fit to a single-site binding equation in GraphPad Prism to calculate the dissociation constant, K d .

Analysis of chemical cross-linking of [2-17]Arf1 to PH ASAP1
20 M [2-17]Arf1-TAMRA and 20 M PH ASAP1 were mixed in the presence of 500 M sucrose-loaded LUV with 2.5% PIP 2 and 15% PS in 100 mM Mes, pH 6.0, 150 mM NaCl. After 20 min, EDC was added to a final concentration of 600 M. The mixture was incubated for 2 h at 25°C with shaking. The reaction was quenched with 4 mM ␤-mercaptoethanol. The pH of the reaction was adjusted to 7 by addition of Tris, pH 8. The sample was treated with trypsin followed by centrifugation to precipitate the LUVs. Mass spectra of the trypsin-treated supernatants were collected with an Orbitrap Fusion mass spectrometer, and data were analyzed with pLink software (87,88).

Preparation of membrane scaffolding protein (MSP) belt proteins
The plasmids for MSP⌬H5 were the generous gift of Drs. Franz Hagn and Gerhard Wagner (Harvard Medical School) to Dr. Byrd. Theproteinswereexpressedandpurifiedasdescribedpreviously (89).

NMR measurements and chemical shift perturbation analysis
All NMR spectra were collected at 25°C on a Bruker Avance III 850 MHz spectrometer equipped with TCI triple-resonance cryoprobes. Chemical shift perturbations were analyzed by Equation 3, Ϫ 4 ⅐ ͑n ⅐ P t ͒ ⅐ ͑L t ͔͒ 1/ 2 ]/ 2 ⅐ n ⅐ P t (Eq. 3) where ⌬␦ obs , ⌬␦ max , P t , L t , and k d values are the change in the observed chemical shift from the free state, the maximum change in chemical shift, the total PH domain concentration, the concentration of ND, and the dissociation constant, respectively. Data converged to n, the number of sites, equal to 2, indicating that a nanodisc can bind one PH domain on each of its sides.

Cell biology
NIH 3T3 clone 7, kindly provided by Dr. Douglas Lowy, was maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1ϫ penicillin/streptomycin. Cells were transfected with 2.5 g/ml plasmids for expression of GFP or the indicated ASAP1 protein fused to GFP with Lipofectamine LTX Plus reagent and used 24 h later. CDRs were induced in cells plated on 10 g/ml fibronectin-coated coverslips that were maintained for 5 h in Opti-MEM with no serum by treating with 20 ng/ml PDGF for 5 min. Cells were fixed with 4% paraformaldehyde. Before staining for immunofluorescence, cells were permeabilized with 0.2% saponin, 0.5% BSA, and 1% fetal bovine serum in PBS. Actin in the CDRs was stained with Alexa 594 phalloidin, and ASAP1-GFP or GFP was visualized using primary GFP rabbit polyclonal antibody followed by the anti-rabbit secondary antibody conjugated to Alexa Fluor 488. Mounting was done using Dako fluorescence mounting medium. Microscope images were captured using a Leica SP8 laser-scanning confocal microscope using a ϫ20, 0.75 numerical aperture objective (Leica Microsystems Inc, Buffalo Grove, IL). Single slices were collected with the pinhole size set to 6.28 airy units. At least 25 cells were counted for each condition for each of four experiments (two for [R497K]ASAP1).