Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3′-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3Kα, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3Kα in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3Kα. Our results revealed that Lys-776, located in the P-loop of PI3Kα, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3Kα autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3Kα kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3Kα. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3Kα catalysis.

PIK3CA, the gene that encodes the catalytic subunit of PI3K␣, is somatically mutated in diverse tumor types. The majority of mutations in solid tumors (Ͼ80%) are in the conserved regions of the helical and kinase domains (7), most commonly E542K and E545R in the helical domain and H1047R in the kinase domain (17). These mutations result in PI3K␣ enzymes with increased phosphoinositide kinase activity (18 -21), leading to up-regulation of downstream signaling events that modulate the function of numerous substrates involved in the regulation of cell survival, cell cycle progression, and cellular growth (18).
In addition to its lipid kinase activity, PI3K␣ displays a protein kinase activity that results in the autophosphorylation of the p85␣ subunit (22,23). The effect of this autophosphorylation is controversial; although in some studies it is reported that autophosphorylation of Ser-608 of p85␣ inhibits the PI3K␣ lipid kinase activity (23)(24)(25), at least one study reported that autophosphorylation did not affect the lipid kinase activity (26). Differences in the preparation of the protein may account for the conflicting results; the latter work used recombinant protein, whereas the former used immunoprecipitated enzyme. In contrast to the vast knowledge of the biological significance of PI3K␣ lipid kinase activity in cell signaling, the physiological relevance of the protein kinase activity of PI3K␣ is still not well characterized (27,28).
Structural studies on PI3Ks have revealed many features of the kinase domain including the presence of the phosphatebinding loop/P-loop and of the catalytic and activation loops described in the well-characterized protein kinases (29,30). In the previously published structure of PI3K␣ with diC4-PIP 2 (PDB ID 4OVV), we determined that the PIP 2 substrate binds in a groove formed by the P-loop (p110␣ residues 772-778), the activation loop of the kinase domain (p110␣ residues 935-958), and the iSH2 coiled-coil (31) (Fig. 1B). Modeling of bound ATP shows that the 3Ј hydroxyl group of diC4-PIP 2 is oriented toward the ␥-phosphate of the ATP (Fig. 1B). Furthermore, the short-chain lipid substrate is in a position accessible to the membrane, which would allow the C-18 and C-20 hydrophobic tails of the physiological PIP 2 substrate to be buried into the membrane. Being negatively charged, its placement in the structure makes it well-suited to interact with the basic residues on the surface of the iSH2 domain and those of the loops of the C2 and kinase domains as originally proposed in a model of the lipid membrane interaction with the p110␣/niSH2 heterodimer (30). Despite the favorable orientation of the PIP 2 substrate, the structure of this complex represents an inactive conformation of PI3K␣, as the reactive 3-OH of the PIP 2 is too far (ϳ6 Å) from the ␥-phosphate of the ATP for a productive phosphoryl transfer. An additional characteristic suggesting that this is an inactive enzyme conformation is a salt bridge between Lys-948 of the activation loop and Glu-342 of the nSH2 domain, which locks the kinase domain and prevents the nSH2 domain from binding to effectors (31). However, unlike in most of the previous p110 structures, in the PI3K␣-PIP 2 complex structure the activation loop is ordered; it is nested near the iSH2 and nSH2 domains of p85␣ and positioned in a way that allows communication between p85␣ and the kinase domain of p110␣.
Previous biochemical studies on PI3Ks have reported that Lys-802 in p110␣ (Lys-833 in p110␥) is involved in phosphate transfer reaction (29,32). It was also shown that the activation loop of p110␣ is a determinant of lipid substrate specificity and not protein kinase activity and that Lys-942 and Lys-949 within the activation loop of p110␣ (Lys-973 and Ar-980, respectively, in the activation loop of p110␥) are required for lipid substrate recognition (33). Furthermore, in the conserved DRHXXN motif of the catalytic loop, Asp-731 and Asn-736 of Vps34p (Class III PI3K) and Arg-916 of p110␣ are essential for the kinase activity of PI3Ks (23,34).
Despite the wealth of information on the PI3K␣ active site, the identity and role of many of the individual residues in lipid and protein phosphorylation remain largely unknown. To answer these fundamental questions, we used the structure of the PI3K␣-PIP 2 complex as a template and selected residues that lie within 9 Å of the catalytic PIP 2 -binding site for analyzing the effect of substitutions on these residues. Six p110␣ mutants, H917A-p110␣/p85␣, H936A-p110␣/p85␣, K776E-p110␣/p85␣, R777E-p110␣/p85␣, E722A-p110␣/p85␣, and

Lys-776, His-936, and His-917 are crucial for the lipid kinase activity of PI3K␣
The kinetics of the lipid kinase activity of WT PI3K␣ and the mutants were studied using a fluorescence polarization assay. The K m of WT PI3K␣ was 1.77 Ϯ 0.03 M for PIP 2 and 2.0 Ϯ 0.5 M for ATP with a V max of 1.78 Ϯ 0.06 pmol/min (Table 1). Of the eight PI3K␣ mutants, three showed a dramatic change in their kinetic parameters when compared with those of the WT enzyme (Table 1, Fig. 2, C and D). K776E-p110␣/p85␣ showed a very high K m for both ATP and PIP 2 substrates (Table 1), suggesting a role of Lys-776 in the recognition of both substrates. H917A-p110␣/p85␣ and H936A-p110␣/p85␣ of the catalytic and activation loops, respectively, showed a ϳ50 -100ϫ reduction in their maximal activity compared with WT PI3K␣ (Table 1), indicating that these residues play a role in catalysis. In the structure of the PI3K␣ complex with PIP 2 , these two histidine residues do not make a direct interaction with either of the substrates and are too far to act as catalytic bases, but conformational changes during the catalytic cycle may

Mechanistic insights into PI3K␣
bring these residues closer to a catalytically competent position as has been shown for protein kinases (35,36).
The kinetic parameters of the other PI3K␣ mutants (E722A-p110␣/p85␣, K724E-p110␣/p85␣, R777E-p110␣/p85␣, p110␣/ R461E-p85␣, and p110␣/R465E-p85␣) did not change significantly from those of the WT PI3K␣. Glu-722 of p110␣, located on the N-lobe, is close to a second PIP 2 -binding site identified in the PI3K␣-PIP 2 complex structure (31). Although kinetic analysis shows that it is not involved in the recognition of the catalytic PIP 2 , this assay, performed with soluble PIP 2 , does not rule out that it may interact with the second lipid-binding site, as seen in the crystal structure. If, as suggested, this second site aids in anchoring PI3K␣ to the plasma membrane, this mutation may have an effect on the in vivo activity. Lys-724 of p110␣, also present on the N-lobe, is closer than Glu-722 to the PIP 2 in the active site of the enzyme, and being positively charged, it could have a role in stabilizing the negative charge of PIP 2 ; however, the kinetic analysis presented here suggests that it does not. Arg-777 in the P-loop of p110␣ is not involved in substrate recognition, in contrast to its neighboring residue, Lys-776. The lack of conservation of this arginine in the P-loop of protein kinases suggests that it does not play a role in the recognition of ATP. On the other hand, the lack of effect on the catalytic activity of the p85␣ mutants (p110␣/R461E-p85␣ and p110␣/R465E-p85␣) argues against a role of these arginine residues in compensating the negative charge of the phosphates at positions 4 and 5 on the inositol ring of PIP 2 but does not rule out the possibility of their participation in hydrogen bonding to PIP 2 .

Bisubstrate kinetic analysis of PI3K␣ indicates an ordered sequential mechanism
The kinetic mechanism of the wild-type enzyme was elucidated with initial velocity studies analyzed using double-reciprocal plots. Lineweaver-Burk analysis of velocities with varying concentrations of ATP at different fixed concentrations of PIP 2 displayed convergence of a set of lines in the second quadrant (Fig. 3A). A similar analysis of rate data with varying concentrations of PIP 2 at different fixed concentrations of ATP yielded a set of lines intersecting on the 1/v axis (Fig. 3B). This pattern of intersecting lines on the vertical axis is indicative of an ordered sequential mechanism involving the formation of a ternary complex (37). The dissociation constants, K a for ATP and K b for PIP 2 , were determined from the global fit of the data by nonlinear regression analysis to the ordered bisubstrate kinetic model. The dissociation constants K a and K b for the WT PI3K␣ were 10.45 Ϯ 0.25 M and 2.49 Ϯ 0.21 M, respectively. The values of K a and K b for the K776E-p110␣/p85␣ mutant were 146.23 Ϯ 2.62 M and 11.0 Ϯ 0.01 M, respectively. The high dissociation constants for K776E-p110␣/p85␣ mutant compared with the wild-type enzyme are in agreement with the low affinity of this mutant for both the substrates, as observed in the lipid kinase assays described above.

Lys-776 and His-917 are critical for autophosphorylation
In addition to PIP 2 phosphorylation, the catalytic subunit of PI3K␣, p110␣, phosphorylates Ser-608 of its regulatory subunit, p85␣. This phosphorylation has been proposed to have a regulatory effect on the activity of PI3K (23). To assess autophosphorylation, we used radioactive ␥-32 P-labeled ATP in the PI3K␣ kinase reaction. We separated the proteins by SDS-PAGE and visualized the radioactive bands by autoradiography. The reaction carried out with WT PI3K␣ displayed a strong signal for p85␣, clearly indicating its autophosphorylation (Fig.  4A). On the other hand, the PI3K␣ mutants H917A-p110␣/ p85␣ and K776E-p110␣/p85␣ lack protein kinase activity (Fig.  4A). These results are consistent with our lipid kinase assays that showed H917A-p110␣/p85␣ was inactive for lipid kinase activity and K776E-p110␣/p85␣ showed a very low-binding affinity for ATP. The other mutants (H936A-p110␣/p85␣, R777E-p110␣/p85␣, E722A-p110␣/p85␣, and K724E-p110␣/ p85␣) displayed a signal for p85␣ phosphorylation comparable with that of the WT, suggesting that these residues do not play a role in the substrate-binding and catalytic mechanism of protein kinase activity of PI3K␣. Interestingly, in the presence of lipid substrate PIP 2 , the autophosphorylation activity of the wild-type enzyme was almost negligible compared with the reaction in the absence of PIP 2 (Fig. 4B). The high PIP 2 concentration (50 M) may compete with the protein substrate for the catalytic site of the enzyme, suggesting that the PIP 2 substrate site and the protein substrate site overlap at least partially.

Cellular phosphorylation of WT PI3K␣ and mutants
To assess the extent of phosphorylation of Ser-608 of the p85␣ subunit of PI3K␣ in cells, we purified the enzymes from infected Sf9 insect cells and mapped the phosphorylated sites in the recombinant WT PI3K␣ enzyme and mutants by phosphoamino acid analysis using tandem mass tag (TMT) mass spectrometry. For this, we differentially labeled WT PI3K␣ and mutants with isobaric tandem mass tags that release specific reporter ions upon fragmentation. Reporter ion intensities obtained by MS/MS were used to quantify the phosphorylated peptides in WT PI3K␣ and mutants. We compared the ratios of phosphorylated to unphosphorylated Ser-608 for the WT and mutants. Three mutants (H936A-p110␣/p85␣, K776E-p110␣/ p85␣, and H917A-p110␣/p85␣) showed a remarkable decrease in phosphorylation of Ser-608 compared with the WT enzyme (Fig. 4C). The phosphorylation levels of Ser-608 in K776E-p110␣/p85␣ and H917A-p110␣/p85␣ in insect cells are in agreement with our in vitro lipid and protein kinase assays which show that these mutants have highly impaired enzymatic activity.
Furthermore, MS/MS analysis of WT PI3K␣ showed that the p85␣ subunit is phosphorylated at Ser-43, Ser-154, Ser-208, and Thr-603 in addition to Ser-608. In the available PI3K␣ structure, the positions of these residues are not within the reach of the catalytic site, suggesting that these phosphorylations most likely are due to the catalytic action of other endogenous protein kinases present during recombinant PI3K␣ expression in insect cells.

Discussion
The structure of the complex of PI3K␣ (harboring p110␣ and nip85␣) with PIP 2 most likely reflects a nonproductive binding mode and does not provide clear candidates for residues that participate in catalysis. To close this gap in knowledge, we cast a wide net to identify possible key residues and selected 8 residues within 9 Å of the PIP 2 -binding site for further studies. The

Mechanistic insights into PI3K␣
rationale behind selecting residues at distances as large as 9 Å from the substrates was based on the assumption that closing of the active site upon ATP binding, as described in protein kinases, would bring the substrates, ATP and PIP 2 , closer to each other and would also position the catalytic residues to facilitate transfer of the phosphoryl group (38). Furthermore, the phosphate tail of the ATP is flexible and any small conformational change in the active site could bring the ␥-phosphate of the ATP much closer to the PIP 2. and form an active enzyme complex for catalysis. Using mutational and kinetic analysis, we identified three p110␣ residues that are essential for catalytic activity: Lys-776, His-917, and His-936.
We determined that Lys-776 in the P-loop of p110␣ is involved in the recognition of ATP as well as PIP 2 . This residue is also crucial for autophosphorylation of the p85␣ subunit. Despite the low sequence conservation of the P-loop, its threedimensional structure is conserved in most mononucleotidebinding proteins, including protein kinase families. In most of the ATP-and GTP-binding proteins, a conserved lysine residue in the P-loop is implicated in binding to the ␥-phosphate of NTPs (39,40). Furthermore, in the crystal structure of PI3K␣ in complex with PIP 2 and with ATP modeled, the P-loop (residues 772-778) shows a conformation similar to that found in other P-loop-containing proteins, with Lys-776 oriented toward the ␤ and ␥ phosphates of ATP (Fig. 5, A and B). The side chain of this lysine faces the 1-phospho group of PIP 2 and may form a water-mediated hydrogen bond (31). This arrangement points toward the role of this lysine in phosphoinositide interaction (Fig. 5, A and B) even though it might not be able discriminate between different phosphoinositides. These observations are in agreement with the large effect of the K776E mutation on the K m of the enzyme for both the substrates. The charge reversal mutation K776E was selected to investigate whether this lysine is required to compensate the negative charge of ATP and/or PIP 2 , as hypothesized by Pirola et al. (33). The K776E mutation would amplify the effect of any charge-dependent interaction, making it easier to detect. Because both the substrates are highly negatively charged, a charge reversal in the active site might result in a loss of activity regardless of the function of this residue. However, our results from a similar charge reversal mutation on the neighboring residue, R777E, did not show any change in the enzyme activity and substrate affinity. This suggests that the results from the K776E mutation are indeed reflective of a role in substrate-binding rather than a nonspecific effect. Structurally, Lys-776 is not close enough to either of the substrates to interact with them; however, conformational changes in the N-and C-lobes and in the flexible P-loop region upon ATP-binding could bring this lysine closer to the two substrates.
Our data show that His-936, located in the activation loop of PI3K␣, is critical for lipid kinase activity but not required for protein phosphorylation. In the p110␣ sequence, His-936 is present immediately after the conserved DFG motif (Fig. 5C). All protein kinases known contain a conserved DFG sequence (Mg 2ϩ -orienting loop) in the activation segment of their catalytic domains, and this motif has a critical function in catalysis (41). His-936 is conserved in all class I PI3Ks but not in protein kinases (Fig. 5C), suggesting that this histidine is required for lipid phosphorylation and not for the protein kinase activity. Moreover, His-936 is in the vicinity of the 3Ј hydroxyl on the inositol ring of PIP 2 , but is relatively far from the serine hydroxyl group on the peptide substrate modeled in our p110␣/ nip85␣ structure (Fig. 5B). On the basis of this structural obser- Shown is the structure of the kinase domain of p110␣ harboring the P-loop and catalytic and activation loops with the lipid substrate (A) and the peptide substrate modeled from a cyclin-dependent protein kinase (CDK2) structure (B) (PDB ID 1QMZ). Lys-168 of PKA, shown in magenta, aligns with His-917 of p110␣. C, sequence alignments of the catalytic loop (residues 912-920) and activation loop (residues 935-958) segments of class IA isoforms of PI3K (␣, ␥, ␦) with different protein kinases (c-Src, tyrosine kinase; PHK, phosphorylase kinase; CDK2, cyclin-dependent kinase 2; PKA, protein kinase A). The conserved DFG motif of the activation loop and the XXDRH/HRDXK motif of the catalytic loop are highlighted in blue and purple, respectively. Mutated histidine residues His-917 and His-936 are marked with stars. D, molecular contacts between the substrates, conserved active-site residues, and the Mg 2ϩ ions are shown. Asp-933 of the conserved DFG motif in the activation loop binds to the Mg 2ϩ ion to orient the ␥-phosphate of ATP for transfer. Asn-920 interacts with a second Mg 2ϩ ion, which chelates the ␤-phosphate of ATP. Lys-776 of the conserved P-loop may support interactions with the ␥-phosphate of ATP as well as 3Ј-OH of PIP 2 . His-936 of the activation loop is proposed to act as the catalytic base. His-917 of the conserved XXDRH motif in the catalytic loop, interacts with the ␥-phosphate of ATP, and helps in the phosphoryl transfer step.

Mechanistic insights into PI3K␣
vation, we propose that His-936 may act as a catalytic base to deprotonate the 3Ј hydroxyl of the PIP 2 substrate and promote the nucleophilic attack on the ␥-phosphate of ATP. Although this histidine is not close enough to the 3Ј-OH of PIP 2 to deprotonate it, it is possible, as observed in protein kinases, that a conformational change involving the N-and C-lobes could bring His-936, present on the flexible activation loop, closer to PIP 2 . In addition, physiological activation of PI3K␣, triggered by the binding of phosphorylated proteins to the nSH2 domain of the regulatory subunit, could release the network of hydrogen bonds between the activation loop and the nSH2 domain (31,42) and allow the activation loop bearing the catalytic His-936 to adopt an active conformation in closer proximity to the lipid substrate.
His-917, located on the catalytic loop, is required for the lipid as well as protein kinase activities of PI3K␣ as shown by the lack of activity of the H917A-p110␣/p85␣ mutant in this study. His-917 is part of the conserved XXDRH motif in the catalytic loop of PI3Ks. In contrast, the catalytic loop of protein kinases contains a conserved HRDXK motif at the corresponding position in the sequence (Fig. 5C). Sequence as well as structural alignment of PI3K␣ with a prototypical protein kinase PKA (cAMPdependent protein kinase) shows that the lysine (Lys-168) in the HRDXK motif of PKA is at the position of the PI3K XXDRH histidine (Fig. 5, B and C). The lysine of the HRDXK motif is conserved in all Ser/Thr protein kinases and is replaced by an arginine in tyrosine protein kinases (43,44). In light of these facts, we propose that His-917 of PI3K␣, due to its proximity to ATP, may interact electrostatically with the ␥-phosphate of ATP and mediate phosphoryl transfer as reported for the corresponding Lys-168 of PKA (45,46).
The HRDXK histidine of protein kinases has been described as one of the residues of the R-spine (Rϭ regulatory). R-spine is a highly conserved spatial motif consisting of four non-consecutive residues that connects the two lobes of kinases and is found only in the active protein kinases (36). Disruption of R-spine affects the enzyme activity of protein kinases, suggesting a regulatory function (47). However, structural alignment of PI3K␣ with protein kinases does not show a similar spatial arrangement of residues characteristic of the R-spine of protein kinases. This suggests that for PI3Ks, the residues of the R-spine should be redefined. Furthermore, recent studies in Aurora kinases reveal the involvement of the HRDXK histidine in preserving the active conformation of the DFG motif through a hydrophobic interaction between this histidine and the DFG phenylalanine (48). This arrangement defines another important role for the HRDXK histidine in protein kinases. However, in the PI3K␣ structure, the arginine of the conserved XXDRH motif seems to play a role in the interaction with the DFG motif (Fig. 5A).
The lipid kinase mechanism of PI3K␣ that emerges from the kinetic and structural analyses involves a nucleophilic attack by the 3Ј-OH of the inositol on the ␥-phosphate of ATP (Fig. 5D). His-936, being closer to the inositol ring of the lipid substrate, is the catalytic base required for the deprotonation of the 3Ј hydroxyl. Two Mg 2ϩ ions and Lys-776 of the P-loop compensate the charge of the bound ATP (Fig. 5D). Furthermore, the proximity of Lys-776 to the 3Ј-OH of the PIP 2 substrate aids the reaction by lowering the hydroxyl pK a . His-917 of the catalytic loop is in a position to make an electrostatic interaction with the ␥-phosphate of ATP and help in phosphotransfer (Fig. 5D). The proposed roles of Lys-776, His-936, and His-917 in this mechanism provide a rationale for the observed effects of their mutations in lipid and protein kinase activities of PI3K␣. Besides elucidating substrate specificity and catalytic mechanism, our studies highlight the kinetic mechanism of PI3K␣. We performed classic bisubstrate steady-state kinetic analysis of PI3K␣ that suggests a ternary complex mechanism involving direct phosphoryl transfer from ATP to PIP 2 .
Taken together, our studies report the first detailed kinetic analysis of PI3K␣ active-site residues that resulted in the identification of three residues critical for PI3K␣ catalysis and allowed us to propose a catalytic mechanism. In addition, we showed similarities and differences between the lipid and protein kinase specificities of PI3K␣. Our data suggest that the lipid and protein kinase activities are mediated in part by different structural motifs in the kinase domain of PI3K␣. Moreover, we analyzed for the first time the bisubstrate kinetic mechanism of the PI3K␣ lipid kinase activity. These findings constitute a significant advance in the characterization of dual kinase specificity of PI3K␣, a key enzyme in the AKT/mTOR signaling pathway that regulates diverse cellular processes like survival, proliferation, and motility as well as protein synthesis and vesicle trafficking. The identification of PI3K␣ residues involved in substrate recognition and catalysis will have a significant impact in many fields and disciplines ranging from the regulation of glucose homeostasis to the design of cancer chemotherapeutic agents.

Purification of WT PI3K␣ and mutants
Cell pellets harvested from a 4-liter culture of Sf9 cells co-infected with p110␣ and p85␣ recombinant baculoviruses were resuspended in 250 ml of resuspension buffer (50 mM sodium phosphate, pH 8.0, 400 mM NaCl, 10 mM imidazole, 1% Triton X-100, 5% glycerol, 1 mM sodium orthovanadate, 5 tablets of Complete Protease Inhibitor Mixture (Roche Applied Science)) and lysed by sonication. The soluble supernatant was loaded onto nickel-nitrilotriacetic acid beads (Qiagen). The complex of p110␣ and p85␣ was eluted with 250 mM imidazole. The nickel-nitrilotriacetic acid elutes containing both the subunits were pooled and desalted using HiPrep 26/10 desalting column (GE Healthcare). The protein was further purified by anionexchange chromatography using a Mono Q column (GE Healthcare). PI3K␣ was eluted using a linear gradient of 0 -500 mM NaCl. The anion-exchange fractions containing pure PI3K␣ were pooled, and the protein concentration was determined by the Bradford assay (Bio-Rad). All PI3K␣ mutants used in the study (six p110␣ mutants and two p85␣ mutants) were purified by the same protocol. A Superose 6 (10/300 GL) gelfiltration column was used to determine and compare the oligomeric state of WT PI3K␣ and mutants. The native molecular weight of WT PI3K␣ was determined from a standard curve for this column.

Fluorescence polarization activity assay for PI3K␣
To characterize the lipid kinase activity of WT PI3K␣ and its mutants, we used a fluorescence polarization assay (Echelon Biosciences).

Bisubstrate kinetics
Initial velocity studies of PI3K␣ were performed using the fluorescence polarization assay described above to determine both the bisubstrate kinetic mechanism of WT PI3K␣ and the dissociation constants of the reaction. Double-reciprocal plots were plotted for velocities versus substrate concentrations by varying ATP concentration from 5 to 15 M at different fixed non-saturating concentrations of PIP 2 (5, 7.5, 10, and 15 M). The data were fitted to bisubstrate sequential equations using SigmaPlot 13, and the dissociation constants K a for ATP and K b for PIP 2 were calculated by nonlinear regression analysis. Equations for ordered (Equation 1) and random bi-bi mechanisms (Equation 2) are listed below, respectively (49).

Radioactive PI3K␣ protein kinase assay
We used a radioactive-based protein kinase assay to analyze the autophosphorylation of the p85␣ subunit of WT PI3K␣ and mutants. The reaction mixture contained 20 mM HEPES, pH 7.5, 10 mM MgCl 2 , 0.25 mM DTT, 1 g of PI3K␣, and 1 l of ␥-32 P-labeled ATP (3000 Ci/mmol, 10 mCi/ml, PerkinElmer Life Sciences) at a final concentration of 10 M ATP in a total of 50 l. To assess the autophosphorylation in the presence and absence of lipid substrate, 50 M PIP 2 was added to the reaction mixture. The incubation was done at 30°C for 30 min. Samples were separated by 8 -16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylated products were visualized by autoradiography.

Phosphorylation analysis and quantification by mass spectrometry
WT PI3K␣ and six p110␣ mutants were identified for phosphorylation sites by TMT mass spectrometry. For this, samples were prepared by denaturation, reduction, alkylation, and tryptic digestion. Next, samples were differentially labeled with isobaric mass tags (TMT 10-plex, ThermoScientific) according to the manufacturer's specifications. All the TMT-labeled peptides were mixed and fractionated by basic reverse phase chromatography. Each fraction was analyzed by tandem mass spectrometry (MS/MS), and data analysis was performed. Peptide and fragment ion masses were extracted from the raw mass spectra in Proteome Discoverer (PD) software (v1.4, Thermo-Scientific) and searched using Mascot (v2.5.1) against a custom database containing the wild-type proteins and their respective mutants. The reporter ions from all peptides were quantilenormalized to minimize any technical variation in sample preparation and TMT labeling. For quantification, the intensities of seven reporter ions obtained by MS/MS were used to compare the relative amounts of phosphorylated to unphosphorylated peptides in all seven samples, and the results were reported as ratios of phosphorylated to unphosphorylated peptides for WT PI3K␣ and its mutants.