Diversification of cardiac insulin signaling involves the p85 alpha/beta subunits of phosphatidylinositol 3-kinase

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 alpha and p85 beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 +/- 0.7-fold over basal) of p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.     

The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance

Insulin resistance is a defining feature of type 2 diabetes and the metabolic syndrome. While the molecular mechanisms of insulin resistance are multiple, recent evidence suggests that attenuation of insulin signaling by c-Jun N-terminal kinase (JNK) may be a central part of the pathobiology of insulin resistance. Here we demonstrate that the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI3K), a key mediator of insulin's metabolic actions, is also required for the activation of JNK in states of insulin resistance, including high-fat diet-induced obesity and JNK1 overexpression. The requirement of the p85alpha regulatory subunit for JNK occurs independently of its role as a component of the PI3K heterodimer and occurs only in response to specific stimuli, namely, insulin and tunicamycin, a chemical that induces endoplasmic reticulum stress. We further show that insulin and p85 activate JNK by via cdc42 and MKK4. The activation of this cdc42/JNK pathway requires both an intact N terminus and functional SH2 domains within the C terminus of the p85alpha regulatory subunit. Thus, p85alpha plays a dual role in regulating insulin sensitivity and may mediate cross talk between the PI3K and stress kinase pathways.     

Positive and negative regulation of phosphoinositide 3-kinase-dependent signaling pathways by three different gene products of the p85alpha regulatory subunit

Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85alpha regulatory subunit yields three splicing variants, p85alpha, AS53/p55alpha, and p50alpha. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110alpha catalytic subunit. PI 3-kinase activity associated with p50alpha was greater than that associated with p85alpha or AS53. Increasing the level of p85alpha or AS53, but not p50alpha, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85alpha mutant lacking the p110-binding site (Deltap85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70(S6K)), was decreased in cells expressing p85alpha or AS53 but not in cells expressing p50alpha. Similar inhibition of PI 3-kinase, Akt, and p70(S6K) was observed, even when p110alpha was coexpressed with p85alpha or AS53. Expression of p110alpha alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110alpha was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.     

The p85alpha regulatory subunit of class IA phosphoinositide 3-kinase regulates beta-selection in thymocyte development

We examined the role of class IA PI3K in pre-TCR controlled beta-selection and TCR-controlled positive/negative selection in thymic development. Using mice deficient for p85alpha, a major regulatory subunit of the class IA PI3K family, the role of class IA PI3K in beta-selection was examined by injection of anti-CD3epsilon mAb into p85alpha(-/-)Rag-2(-/-) mice, which mimics pre-TCR signals. Transition of CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes triggered by anti-CD3epsilon mAb was significantly impaired in p85alpha(-/-)Rag-2(-/-) compared with p85alpha(+/-)Rag-2(-/-) mice. Furthermore, DP cell numbers were lower in p85alpha(-/-)DO11.10/Rag-2(-/-) TCR-transgenic mice than in DO11.10/Rag-2(-/-) mice. In addition, inhibition by IC87114 of the major class IA PI3K catalytic subunit expressed in lymphocytes, p110delta, blocked transition of DN to DP cells in embryonic day 14.5 fetal thymic organ culture without affecting cell viability. In the absence of phosphatase and tensin homolog deleted on chromosome 10, where class IA PI3K signals would be amplified, the DN to DP transition was accelerated. In contrast, neither positive nor negative selection in Rag-2(-/-)TCR-transgenic mice was perturbed by the lack of p85alpha. These findings establish an important function of class IA PI3K in the pre-TCR-controlled developmental transition of DN to DP thymocytes.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.     

p50alpha/p55alpha phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity

Class Ia phosphoinositide (PI) 3-kinases are heterodimers composed of a regulatory and a catalytic subunit and are essential for the metabolic actions of insulin. In addition to p85alpha and p85beta, insulin-sensitive tissues such as fat, muscle, and liver express the splice variants of the pik3r1 gene, p50alpha and p55alpha. To define the role of these variants, we have created mice with a deletion of p50alpha and p55alpha by using homologous recombination. These mice are viable, grow normally, and maintain normal blood glucose levels but have lower fasting insulin levels. Results of an insulin tolerance test indicate that p50alpha/p55alpha knockout mice have enhanced insulin sensitivity in vivo, and there is an increase in insulin-stimulated glucose transport in isolated extensor digitorum longus muscle tissues and adipocytes. In muscle, loss of p50alpha/p55alpha results in reduced levels of insulin-stimulated insulin receptor substrate 1 (IRS-1) and phosphotyrosine-associated PI 3-kinase but enhanced levels of IRS-2-associated PI 3-kinase and Akt activation, whereas in adipocytes levels of both insulin-stimulated PI 3-kinase and Akt are unchanged. Despite this, adipocytes of the knockout mice are smaller and have increased glucose uptake with altered glucose metabolic pathways. When treated with gold thioglucose, p50alpha/p55alpha knockout mice become hyperphagic like their wild-type littermates. However, they accumulate less fat and become mildly less hyperglycemic and markedly less hyperinsulinemic. Taken together, these data indicate that p50alpha and p55alpha play an important role in insulin signaling and action, especially in lipid and glucose metabolism.     

The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex

Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.     

Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.     

Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase

The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85alpha both in vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85alpha. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric.     

Mechanism of constitutive phosphoinositide 3-kinase activation by oncogenic mutants of the p85 regulatory subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.     

The tyrosine phosphatase CD148 interacts with the p85 regulatory subunit of phosphoinositide 3-kinase

CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro. Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity.     

Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.     

The p85 regulatory subunit controls sequential activation of phosphoinositide 3-kinase by Tyr kinases and Ras

Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit that regulates a variety of cell responses, including cell division and survival. PI3K is activated following Tyr kinase stimulation and by Ras. We found that the C-terminal region of p85, including the C-Src homology 2 (C-SH2) domain and part of the inter-SH2 region, protects the p110 catalytic subunit from Ras-induced activation. Although the p110 activity associated with a C-terminal p85 deletion mutant increased significantly in the presence of an active form of Ras, purified wild type p85-p110 was only slightly stimulated by active Ras. Nonetheless, incubation of purified p85-p110 with Tyr-phosphorylated peptides, which mimic the activated platelet-derived growth factor receptor, restored Ras-induced p85-p110 activation. In conclusion, p85 inhibits p110 activation by Ras; this blockage is released by Tyr kinase stimulation, showing that the classical mechanism of class IA PI3K stimulation mediated by Tyr kinases also regulates Ras-induced PI3K activation.     

Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha IIb beta 3 adhesive function in platelets

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110 beta and p110 gamma) have been implicated in G(i)-dependent integrin alpha(IIb)beta(3) regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110 beta is primarily responsible for G(i)-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin alpha(IIb)beta(3) activation in p110 gamma(-/-) platelets was not associated with alterations in the levels of PI(3,4)P(2) or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110 gamma principally regulated integrin alpha(IIb)beta(3) activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110 gamma, led to a major reduction in integrin alpha(IIb)beta(3) activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110 gamma in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 beta and p110 gamma is critical for P2Y(12)/G(i)-dependent integrin alpha(IIb)beta(3) regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.     

Critical roles of the p110 beta subtype of phosphoinositide 3-kinase in lipopolysaccharide-induced Akt activation and negative regulation of nitrite production in RAW 264.7 cells

It has been suggested that PI3K participates in TLR signaling. However, identifying specific roles for individual PI3K subtypes in signaling has remained elusive. In macrophages from the p110gamma(-/-) mouse, LPS-induced phosphorylation of Akt occurred normally despite the fact that the action of anaphylatoxin C5a was impaired markedly. In RAW 264.7 cells expressing short hairpin RNA that targets p110beta, LPS-induced phosphorylation of Akt was significantly attenuated. In contrast, the LPS action was not impaired, but was rather augmented in the p110alpha-deficient cells. Previous pharmacologic studies have suggested that a PI3K-Akt pathway negatively regulates TLR-induced inducible NO synthase expression and cytokine production. In the p110beta-deficient cells, inducible NO synthase expression and IL-12 production upon stimulation by LPS were increased, whereas LPS-induced expression of COX-2 and activation of MAPKs were unaffected. Together, the results suggest a specific function of p110beta in the negative feedback regulation of TLR signaling.     

Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products

The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.