A direct interaction between oncogenic Ha-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependent transformation of epithelial cells

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.     

Membrane-targeted phosphatidylinositol 3-kinase mimics insulin actions and induces a state of cellular insulin resistance.

Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110(CAAX)), and wild-type p110 (p110(WT)) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110(CAAX) exhibited approximately 2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to approximately 4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110(CAAX) fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110(WT) had little or no effect on these downstream effects. Interestingly p110(CAAX) did not activate MAP kinase, despite its stimulation of p21(ras). Surprisingly, p110(CAAX) did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110(CAAX) also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110(CAAX) induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110(CAAX) stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110(CAAX) expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

The catalytic subunit of phosphoinositide 3-kinase: requirements for oncogenicity

The retroviral oncogene p3k (v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110alpha. The v-P3k protein is oncogenic in vivo and in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.     

Cyclin D1 Expression Mediated by Phosphatidylinositol 3-Kinase through mTOR-p70S6K-Independent Signaling in Growth Factor-Stimulated NIH 3T3 Fibroblasts

Phosphatidylinositol (PI) 3-kinase is required for G₁ to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70^S6K. However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. Here we report that the expression of a dominant negative (DN) form of either the p110α catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibited epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogated increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G₁ arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppressed p70^S6K activity throughout the G₁ phase, had little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, was also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. We also found that an enforced expression of wild-type p110 was sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells was strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but was independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 was sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions.     

Effects of oncogenic p110alpha subunit mutations on the lipid kinase activity of phosphoinositide 3-kinase

The PIK3CA gene, encoding the p110alpha catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110alpha, H1047R, E542K and E545K, were shown to potently activate PI3K signalling in human epithelial cells. In the present study, we examine the biochemical activity of the recombinantly purified PI3K oncogenic mutants. The kinetic characterizations of the wt (wild-type) and the three 'hot spot' PI3K mutants show that the mutants all have approx. 2-fold increase in lipid kinase activities. Interestingly, the phosphorylated IRS-1 (insulin receptor substrate-1) protein shows activation of the lipid kinase activity for the wt and H1047R but not E542K and E545K PI3Kalpha, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.     

Oncogenic Mutations of p110α Isoform of PI 3-Kinase Upregulate Its Protein Kinase Activity

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 βc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 βc receptor (βic). We find p110α, p110β and p110γ all phosphorylate βic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards βic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn²⁺ dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110β and p110γ all display significant activity in the presence of Mg²⁺. Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α.     

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.     

Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110α catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110α, p110β, and p110δ, each coexpressed with the regulatory subunit p85α or splice variant p55α. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.     

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.     

p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.     

Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3ca^H1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (β-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling.     

The catalytic class I(A) PI3K isoforms play divergent roles in breast cancer cell migration

Transforming growth factor-β (TGFβ) plays an important role in breast cancer metastasis. Here phosphoinositide 3-kinase (PI3K) signalling was found to play an essential role in the enhanced migration capability of fibroblastoid cells (FibRas) derived from normal mammary epithelial cells (EpH4) by transduction of oncogenic Ras (EpRas) and TGFβ1. While expression of the PI3K isoform p110δ was down-regulated in FibRas cells, there was an increase in the expression of p110α and p110β in the fibroblastoid cells. The PI3K isoform p110β was found to specifically contribute to cell migration in FibRas cells, while p110α contributed to the response in EpH4, EpRas and FibRas cells. Akt, a downstream targets of PI3K signalling, had an inhibitory role in the migration of transformed breast cancer cells, while Rac, Cdc42 and the ribosomal protein S6 kinase (S6K) were necessary for the response. Together our data reveal a novel specific function of the PI3K isoform p110β in the migration of cells transformed by oncogenic H-Ras and TGF-β1.     

Functional differences between two classes of oncogenic mutation in the PIK3CA gene

PIK3CA codes for the p110α isoform of class-IA PI 3-kinase and oncogenic mutations in the helical domain and kinase domain are common in several cancers. We studied the biochemical properties of a common helical domain mutant (E545K) and a common kinase domain mutant (H1047R). Both retain the ability to autophosphorylate Ser608 of p85α and are also inhibited by a range of PI 3-kinase inhibitors (Wortmannin, LY294002, PI-103 and PIK-75) to a similar extent as WT p110α. Both mutants display an increased V_max but while a PDGF derived diphosphotyrosylpeptide caused an increase in V_max for WT p85α/p110α it did not for the E545K variant and actually decreased V_max for the H1047R variant. Further, the E545K mutant was activated by H-Ras whereas the H1047R mutant was not. Together these results suggest helical domain mutants are in a state mimicking activation by growth factors whereas kinase domain mutants mimic the state activated by H-Ras.     

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP₃/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.     

Then Negative Regulation of Phosphoinositide 3-Kinase Signaling by p85 and Its Implication in Cancer

The phosphoinositide 3-kinase (PI3K) signaling pathway critically regulates cell growth and cell survival. Mutations that lead to aberrant activation of this pathway are frequent events in human cancers. Here we discuss some recent studies identifying the mechanisms by which p85, the regulatory subunit of PI3K, negatively regulates PI3K signaling. While necessary for the stability and membrane recruitment of the p110 catalytic subunit of PI3K. p85 represses the basal activity of p110 in the absence of growth factor stimulation. In its unbound, free form, p85 sequesters the adaptor protein IRS-1 and therefore limits the extent of PI3K signaling downstream of the insulin and IGF-1 receptors. These findings lend new insight to how changes in p85 gene dosage or mutations in p85 could lead to the hyper-activation of PI3K and thus contribute towards tumorigenesis.     

YXXM motifs in the PDGF-beta receptor serve dual roles as phosphoinositide 3-kinase binding motifs and tyrosine-based endocytic sorting signals

Phosphoinositide 3-kinases (PI 3-kinases) are important regulators of endocytic trafficking. Previous studies have shown that mutant human platelet-derived growth factor-beta receptors (PDGFR), which contain Phe in place of Tyr at the two p85/p110 PI 3-kinase binding sites (PDGFR-F/F), are defective for both p85 binding and ligand-stimulated degradation. This suggested that p85/p110 regulates PDGFR trafficking. However, more recent work has identified hVPS34, and not p85/p110, as the major PI 3-kinase regulating the movement of receptors through the endosomal system. To reconcile this discrepancy, we hypothesized that YXXM motifs in the PDGFR might play a second role as Tyr-based lysosomal sorting motifs (YXXPhi). To test this, we replaced both YXXM motifs with a motif from LAMP-1, YQTI. This mutant PDGFR (PDGFR-YQTI) still underwent PDGF-stimulated autophosphorylation but did not bind p85. In CHO cells, both wild-type and YQTI receptors showed PDGF-stimulated turnover, whereas F/F receptors did not. In addition, uptake and degradation of cell surface-labeled YXXM and YQTI receptors was fast relative to F/F receptors. We also constructed chimeras containing extracellular and membrane-spanning domains from CD25 (Tac) and cytoplasmic tails containing the YQTI motif, two YXXM motifs, or two mutant FXXM motifs. The YXXM and YQTI chimeras mediated lysosomal delivery of fluorescein isothiocyanate-labeled anti-CD25 antibodies, whereas the F/F chimera was defective. Thus, YQTI motifs can target PDGFR for degradation in the absence of p85/p110 binding, and the p85/p110 binding motifs from PDGFR are sufficient to target Tac chimeras to the lysosome. These data suggest that the YXXM motifs in the PDGFR serve two distinct functions: PI 3-kinase recruitment and lysosomal targeting.