Modification of phosphatidylinositol 3-kinase SH2 domain binding properties by Abl- or Lck-mediated tyrosine phosphorylation at Tyr-688.

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in cotransfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.
Several additional modes of PI3K regulation have been demonstrated, and it is likely that they act in concert to regulate the production of 3-phosphorylated inositol phospholipids in response to a variety of stimuli. The catalytic p110 interacts with activated GTP-bound Ras proteins through a region adjacent to its p85-binding NH 2 terminus (30,31). Active Ras enhances PI3K activity in intact cells (30,31), but some data indicate that Ras also acts downstream of PI3K (32). In T cells, the two p85 isoforms have been shown to undergo phosphorylation on both serine and threonine (33,34). Tyrosine phosphorylation of the p85 subunit has been shown to occur in many different systems, such as in response to platelet-derived growth factor (3), insulin (35), B cell antigen receptor ligation (4), interleukin-2 (36), and in cells transformed by the Bcr-Abl fusion protein-tyrosine kinase (37)(38)(39)(40). The sites of phosphorylation in p85 have been mapped to tyrosines 368, 508, and 607 in insulin-stimulated cells (35), but the physiological function of this phosphorylation has remained unknown. Tyrosine phosphorylation of p85 seems not to be required for the enzymatic activity of PI3K. Instead, tyrosine phosphorylation of p85 has been reported to correlate with the dissociation of PI3K from the activated insulin receptor kinase (41).
We have studied the tyrosine phosphorylation of p85 in hematopoietic cells, and report that phosphorylation occurs at least at Tyr-688 in the C-terminal SH2 domain. This event does not detectably affect the catalytic activity of PI3K per se, but causes a change in the binding properties of the SH2 domain. This change is likely to modify the function of PI3K in intact cells.
Cells-Jurkat T leukemia cells, and their simian leukemia virus 40 large T antigen-expressing variant, J-TAg (kind gift from Dr. M. Karin), were kept at logarithmic growth in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and antibiotics. HL-60/ Bcr-Abl cells were obtained by retroviral transfection of HL-60 promyelocytic leukemia cells with pSR␣MSVp185 bcr-abl tkneo as described elsewhere (37). COS-1 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum.
Transient Transfections-15 ϫ 10 6 J-TAg cells, HL-60, or HL-60/Bcr-Abl were transfected with a total of 10 -20 g of DNA by electroporation at 960 microfarads and 240 V. Typically, cells were transfected with 15 g of HA-tagged p85 construct. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were harvested two days after electroporation. COS-1 cells were transfected by lipofection with 10 g of DNA and grown for 48 h prior to the experiments as described (42, 44 -47).
Immunoprecipitation-These procedures were as reported before (5,42,46,47). All steps were carried out at 0 -4°C. Cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA containing 1% Nonidet P-40, 1 mM Na 3 V0 4 , 10 g/ml aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 100 g/ml soybean trypsin inhibitor and clarified by centrifugation at 13,000 ϫ g for 10 min. The clarified lysates were preabsorbed on agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. The lysates were then incubated with antibody for 2-4 h, followed by agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 M NaCl, again in lysis buffer, and either suspended in SDS sample buffer or washed further for kinase assays.
SDS-PAGE and Western Blotting-Proteins were separated by SDS-PAGE and transferred onto nitrocellulose filters. The antisera were used at 1:500 -1:2000 dilution and the blots developed by the enhanced chemiluminescence technique (ECL kit, Amersham) according to the manufacturer's instructions.
In Vitro Phosphorylation of GST Fusion Proteins-The GST fusion proteins were dissolved in kinase buffer (10 mM HEPES pH 7.5, 0.1% Triton X-100, 20 mM MgCl 2 , 1 mM dithiothreitol, and 0.1 mM Na 3 VO 4 ). After addition of 2 mM cold ATP or 1 M ATP and 10 Ci of [␥-32 P]ATP and 1 l of purified Lck or 100 units of Abl-K, the reaction was carried out for 30 min at 30°C or overnight at room temperature (for higher stoichiometry of phosphorylation). The reaction was stopped by adding SDS sample buffer or by adding cold lysis buffer. In the latter case, the mixture was incubated with glutathione-Sepharose beads for 1 h, and the beads washed extensively before they were added to precleared cellular lysates.
Binding of Cellular Proteins to GST Fusion Proteins-These experiments were done as before (48). Cell lysates were prepared as above, cleared by centrifugation, and preadsorbed to glutathione-Sepharose beads. After removal of the beads, the lysates were incubated on ice with 5 g of GST fusion protein and glutathione-Sepharose beads for 2 h, which were subsequently washed five times with lysis buffer. The bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting as above.
Tryptic Peptide Mapping-GST-p85-NiC protein phosphorylated by Lck in the presence of [␥-32 P]ATP was resolved on 10% SDS gels, transferred onto a nitrocellulose filter, exposed to film, and the correct band excised. The filter piece was blocked and digested with TPCK-treated trypsin as described in detail by Luo and co-workers (50). The resulting phosphopeptides were separated by electrophoresis on cellulose thin layer plates at pH 1.9 for 27 min followed by ascending chromatography in n-butanol/pyridine/acetic acid/water (75:50:15:60), and exposed to film for 35 h.

Tyrosine Phosphorylation of p85 in Bcr-Abl-expressing HL-60
Cells-Bcr-Abl induces transformation of fibroblasts and hematopoietic cells (51)(52)(53)(54) and confers resistance to apoptosis (55). Cells expressing the Bcr-Abl tyrosine kinase contain elevated amounts of proteins phosphorylated on tyrosine residues. At least in some Bcr-Abl expressing cells, such as HL-60 cells transfected with Bcr-Abl (37), a fraction of the p85 subunit is included among these substrates for enhanced tyrosine phosphorylation. When PI3K was immunoprecipitated from parental HL-60 cells with antibodies against the p85 subunit and immunoblotted with anti-Tyr(P) mAbs, no phosphorylation of p85 could be detected (Fig. 1, lane 1), even if the cells were pretreated with 100 M pervanadate to increase intracellular Tyr(P) content (lanes 2 and 3). In contrast, when anti-p85 immunoprecipitates prepared from HL-60 cells stably transfected with Bcr-Abl (HL-60/Bcr-Abl cells) were analyzed in parallel, a sharp, but not very prominent, band at 85 kDa (in addition to several other phosphoproteins) was seen (Fig. 1,  lane 4). Reprobing of the same filter with anti-p85 revealed that all immunoprecipitates contained equal amounts of p85, which co-migrated precisely with the Tyr(P)-containing 85-kDa band in lane 4. This result was obtained in several independent experiments.
Tyrosine Phosphorylation of p85 Does Not Affect PI3K Activity-To investigate whether this low level tyrosine phosphorylation of p85 in HL-60/Bcr-Abl cells has any effect on the catalytic activity of PI3K, we first immunoprecipitated PI3K from resting Jurkat T cells, and phosphorylated the immune com- plex in vitro with recombinant Abl-K, after which half of the sample was assayed for PI3K activity and the other half separated by SDS-PAGE for analysis by immunoblotting. Abl-K was found to strongly phosphorylate p85 (Fig. 2, left), but this phosphorylation had no effect on the catalytic activity of PI3K (Fig. 2, right, lanes 1 and 2). An anti-p85 blot of the same filter confirmed that the samples contained the same amount of PI3K. A comparison of the amount of p85 protein versus Tyr(P) content between Figs. 1 and 2 and several similar experiments, demonstrated that the stoichiometry of tyrosine phosphorylation was considerably higher in vitro than in vivo. Thus, the lower level of tyrosine phosphorylation of p85 in intact cells is even more unlikely to affect the catalytic activity of PI3K.
To further study the effect of in vivo phosphorylation on PI3K activity, we assayed anti-Tyr(P) and anti-p85 immunoprecipitates from wild-type HL-60 and HL-60/Bcr-Abl cells for PI3K activity. The anti-Tyr(P) immunoprecipitates contained 5-fold higher PI3K activity from HL-60/Bcr-Abl cells than from parental HL-60 cells (Fig. 2, right, lanes 3 and 4). However, this increase was accompanied by a similar increase in the amount of p85 protein present in these immunoprecipitates (Fig. 2,  center, lower panel, lanes 3 and 4). Anti-p85 immunoprecipitates from both cell types contained equal amounts of p85 protein (Fig. 2, center, lower panel, lanes 5 and 6), but an anti-Tyr(P) immunoblot of the same filter revealed that p85 from wild-type HL-60 cells was completely unphosphorylated while p85 and several co-immunoprecipitated proteins were heavily tyrosine phosphorylated from Bcr-Abl expressing cells (Fig. 2, center, lanes 5 and 6). Despite this difference, the PI3K activity in these immunoprecipitates was similar in both cases. In some experiments, there was a marginal increase in PI3K activity in anti-p85 immunoprecipitates from Bcr-Abl-expressing cells, probably due to the binding of phosphoproteins to the SH2 domains of p85, as seen in the anti-Tyr(P) blot.
We conclude from these experiments that PI3K activity correlated closely with the amount of p85 protein regardless of its phosphorylation status. Apparently, tyrosine phosphorylation of p85 is not required for enzymatic activity and at the stoichi-ometry that it occurs in intact cells it does not directly affect the enzymatic activity of PI3K to any measurable degree.
Bcr-Abl Phosphorylates p85 in Its SH2 Domains-To identify in which part of the molecule p85 is tyrosine phosphorylated in HL-60/Bcr-Abl cells, we transiently transfected these cells with a set of HA-tagged truncated p85 constructs, which were subsequently immunoprecipitated and analyzed by anti-Tyr(P) immunoblotting. In these experiments (Fig. 3), wild-type p85 was phosphorylated on tyrosine, as was p85 lacking the inter-SH2 domain (p85⌬iSH2) and the two constructs containing both SH2 domains with or without the inter-SH2-region (NiC and NC, respectively). In contrast, the individual SH2 domain proteins (N-SH2 and C-SH2) did not contain detectable Tyr(P) even on very long exposures. All constructs were expressed at comparable levels as judged by anti-HA tag immunoblotting (Fig. 3, lower panel). Since the N-SH2 and C-SH2 proteins together contain the same amino acids as the NC protein, we conclude that the main phosphorylation occurs in one or both of these two domains, but that both are required for phosphorylation in intact HL-60/Bcr-Abl cells. This requirement correlates with the co-immunoprecipitation of several cellular phosphoproteins with all constructs having both SH2 domains (Fig.  3), but not the single SH2 domains. Thus, proper interaction of p85 with other proteins, perhaps including Bcr-Abl itself, is important for efficient phosphorylation.
Activated Lck Phosphorylates p85 in the C-terminal SH2 Domain-We have previously used a transient COS-1 cell transfection system to study potential interactions between protein-tyrosine kinases and the role of phosphorylation sites (42, 44 -47). When full-length p85 or its truncated versions were expressed in these cells together with an activated (56 -58) Y505F-mutated Lck, we observed that all constructs having two SH2 domains were efficiently phosphorylated in these cells ( Fig. 4). Also the C-SH2 protein reacted with anti-Tyr(P), although the reactivity was weaker than that of the p85⌬iSH2 (Fig. 4, lanes 2 and 3) or full-length p85 or NC (not shown). This lower level of phosphorylation was not due to expression, which was comparable (Fig. 4, lower panel). In contrast, the N-SH2 protein was completely unphosphorylated even on the very long exposure shown in lanes 6 -10 of Fig. 4. Co-expression of Bcr-Abl, Bmx, or Jak2 was equally unable to cause tyrosine phosphorylation of the N-SH2 protein, although these proteintyrosine kinases caused some phosphorylation of other p85 constructs (not shown). We conclude again that phosphorylation of p85 is efficient when both SH2 domains are present, but that the C-SH2 domain can become phosphorylated when expressed alone at sufficiently high levels. Thus, the phosphorylation of larger constructs is most likely to mainly occur in the C-terminal SH2 domain.
Receptor-induced Phosphorylation of p85-NC in T Cells-Since Lck-Y505F was very efficient at phosphorylating p85 proteins in co-transfected COS-1 cells, we decided to examine whether endogenous Lck or other protein-tyrosine kinases in T cells could also catalyze this reaction. Triggering of the T cell antigen receptor on T cells leads to a rapid phosphorylation of many cellular proteins on tyrosine residues (59 -63). When the HA-tagged NC construct was expressed in J-TAg cells and immunoprecipitated following a brief stimulation of the cells with an anti-CD3⑀ mAb, we observed by anti-Tyr(P) immunoblotting that the protein was phosphorylated to a low stoichiometry prior to cell activation, but became increasingly phos-phorylated with time (Fig. 5). Peak tyrosine phosphorylation occurred at the 5-min time point. The NC protein was equally immunoprecipitated in each sample (lower panel), and it also co-precipitated a set of cellular phosphoproteins at 36 -38, 70, and 120 kDa. These proteins are similar in size to those that co-immunoprecipitate with endogenous p85 (5,49) or that bind to GST fusion proteins of the two SH2 domains of p85 (see below).
Both Abl and Lck Phosphorylate the C-terminal SH2 Domain of p85 in Vitro-As phosphorylation of the C-SH2 protein was relatively low in intact cells, perhaps due to less efficient recruitment or inappropriate subcellular location, we decided to test if Abl-K and Lck would display specificity for this region of p85 in vitro. GST fusion proteins were prepared of the N-SH2, C-SH2, NC, and NiC constructs, and these proteins (or control GST) were incubated with recombinant Abl-K or purified Lck in the presence of ATP. As shown in Fig. 6, all fusion proteins, except GST-N-SH2, were well and equally phosphorylated. The GST-C-SH2 was close in size to Abl-K making its phosphorylation difficult to appreciate in lane 5, but the size of Lck was larger and the phosphorylation of GST-C-SH2 is easily seen in lane 6. Control GST was very weakly phosphorylated by Abl-K or Lck, indicating that the phosphorylation of the p85 protein occurred in the p85-derived parts. Similar results were obtained in several independent experiments.
Mapping of the Major Phosphorylation Site in the SH2 Domains of p85 to Tyr-688 -To determine how many tyrosine residues were phosphorylated in the C-terminal SH2 domain of p85 in vitro, we phosphorylated the GST-NiC protein by Abl-K in the presence of [␥-32 P]ATP and analyzed the reaction product by tryptic peptide mapping. As shown in the bottom panel of Fig. 6, the protein was phosphorylated at one major site (peptide a), a few minor sites and a variable site (peptide b), which may be derived from the GST part of the fusion protein as peptides with similar mobility can be seen in other in vitro phosphorylated GST fusion proteins (46). A similar map was obtained when GST-NiC was phosphorylated with Lck (not shown). Since the maps contained only one major peptide, we decided to map the site through site-directed mutagenesis of a few plausible tyrosine residues. We generated the Y607F, Y685F, and Y688F mutants of the GST-NiC protein, subjected them to the same in vitro phosphorylation with Abl-K, and analyzed them by tryptic peptide mapping. As can be seen in the bottom of Fig. 6, the Y607F and Y685F mutants were still phosphorylated on peptide a, while this spot was completely missing from the phosphorylated Y688F mutant. We conclude that the major site for Abl-and Lck-mediated phosphorylation of p85-NiC is Tyr-688 in the C-terminal SH2 domain. This suggests that tyrosine phosphorylation of endogenous p85 and of the larger transfected p85 constructs in HL-60/Bcr-Abl, COS-1, or J-TAg cells occurs largely (but not necessarily exclusively) at Tyr-688 in the C-terminal SH2 domain.
Tyrosine Phosphorylation Modifies the Binding Properties of the SH2 Domain-The location of Tyr-688 in the C-terminal SH2 domain prompted us to ask whether this phosphorylation may impact the ligand-binding function of the SH2 domain. As a precedent for this possibility, we recently reported that phosphorylation of the Lck SH2 domain at the equivalent residue in the EF loop, Tyr-192, causes a dramatic decrease in its binding of ligands (47). To test this possibility in the case of the p85 SH2 domains, we incubated the GST-NC protein with or without ATP and Abl-K overnight at room temperature to obtain as high stoichiometry of phosphorylation as possible. Following this incubation, the GST fusion protein was adsorbed to glutathione-Sepharose, washed extensively, and mixed with a precleared lysate of pervanadate-treated Jurkat cells. After 2 h on ice, the Sepharose beads were again washed extensively and bound proteins eluted in SDS sample buffer, resolved on SDS gels, transferred onto nitrocellulose filters, and analyzed by anti-Tyr(P) immunoblotting. These experiments revealed that preincubation of the GST fusion protein with Abl-K and ATP, but not ATP alone, caused several significant changes to the set of cellular phosphoproteins subsequently bound to the SH2 domains of p85 (Fig. 7). In particular, a band at 21-kDa and another at 70 kDa decreased very markedly, while other bands remained unchanged or even increased. An anti-GST blot showed that the loading of GST fusion protein was equal in all samples. The band at ϳ65 kDa contains the GST-NC itself, and its phosphorylation can be seen in lanes 3 and 4. This result was obtained in several independent experiments, two of which are shown in Fig. 7. In other experiments we obtained similar results with lysates from Jurkat cells treated with anti-CD3⑀ instead of pervanadate (not shown). In the other experiment shown, a similar result was obtained with preincubation of GST-NC with Lck (lane 4) in addition to incubation with Abl-K (lane 3).
The size of the 21-and 70-kDa proteins, and their absence in experiments using HL-60 cells (not shown), prompted us to blot the samples also with antibodies specific for TCR and Zap-70 (64), both of which were found to be present, and the binding of both decreased clearly upon tyrosine phosphorylation of the GST-NC protein. In contrast, the c-Cbl protein was also pres-  1, 4, 7, 10, and 13), with recombinant Abl kinase domain (lanes 2, 5, 8, 11, and 14) or with purified Lck (lanes 3, 6, 9, 12, and 15) in the presence of 1 mM ATP. Note that Abl-K migrates very close to GST-C-SH2 and Lck as GST-NC. Middle panel, anti-GST blot of the same filter. Lower panel, tryptic peptide maps of wild-type, Y607F, Y685F, or Y688F mutated GST-NiC phosphorylated by Abl-K in the presence of [␥-32 P]ATP. Peptide a was not seen in maps of GST, but peptide b is variably present in the maps of other GST fusion proteins phosphorylated in vitro. ent, but bound equally well to unphosphorylated as well as tyrosine-phosphorylated GST-NC. Perhaps this protein binds well to the N-terminal SH2 domain, which may not be affected by phosphorylation of the C-terminal SH2 domain. Attempts to perform these experiments with the GST-C-SH2 protein (instead of GST-NC) were not successful due to the low binding of cellular proteins to this isolated domain. DISCUSSION Taken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo (40).
The analysis of SH2 domain function in p85 is complicated by the tandem arrangement of the two SH2 domains, resulting in their cooperative binding to many ligands. Our results indicate that tyrosine phosphorylation of the C-terminal SH2 domain reduced the affinity for some ligands, while the binding of others was unchanged. Two alternative explanations could be envisualized: either tyrosine phosphorylation changed the ligand selection from the classical Tyr(P)-X-X-methionine to something more or less different, or only the C-terminal SH2 domain was inhibited, while the N-terminal SH2 domain remained unchanged. In the latter case, only of those ligands that bind exclusively to the C-terminal SH2 domain or that require simultaneous binding to both domains will bind less strongly. We have previously observed that expression of the HA-tagged p85 proteins in T cells resulted in the co-immunoprecipitation of phospho-TCR only when both SH2 domains were present in the p85 protein. Such a requirement for two SH2 domains would explain why the TCR binds despite not having the optimal Tyr(P)-X-X-methionine (19) motif. Apparently, two Tyr(P)-X-X-leucine motifs in tandem in TCR can bind the two SH2 domains of p85 simultaneously and thereby increase the affinity to physiologically relevant levels. The binding of PI3K to TCR and CD3 subunits (5,20,23), as well as to isolated phosphopeptides derived from these proteins (21), has been reported. The findings reported in the present paper may explain why p85 binding to these receptor subunits is of low stoichiometry when assessed by co-immunoprecipitation.
In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the proteintyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry.
We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (45,47). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (47). A similar change was reported by Stover and co-workers (65) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (66). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells.