In this study, we demonstrate that PI3K-C2α and PI3K-C2β represent two downstream targets of growth factor receptor signaling cascades. Stimulation of A431 cells with EGF resulted in the rapid recruitment of both class II PI3K enzymes to a phosphotyrosine signaling complex containing the EGFR. Interestingly, the kinetics with which the two molecules were recruited differed markedly (Fig. ). PI3K-C2β accumulated rapidly with a time course similar to that previously reported for class I PI3K enzyme heterodimers (13
). Similar kinetics were observed with HEK293 and Cos 7 cells (Fig. ) and fibroblasts stimulated with PDGF (Fig. ). In contrast, while a proportion of PI3K-C2α was present in phosphotyrosine complexes within minutes, this PI3K isozyme accumulated over a much longer period, about 20 to 40 min, following ligand addition. This difference suggests either differential compartmentalization of the class II PI3K isozymes or a difference in their mechanisms of regulation. Recent studies with the EGFR and other ErbB family members have begun to clarify the events that control the endocytic routing of these receptors following ligand addition. Such data demonstrate that the subcellular localization of the activated receptor influences the nature of its downstream signaling events (17
EGF stimulation of PI3K activity has been described for a large number of primary cells and cell lines. Leydig cells, A431 cells which express EGFR to high levels (approximately 106
receptors per cell), and murine fibroblasts transfected with recombinant EGFR display increased PI3K activity in antiphosphotyrosine antibody immune complexes following EGF addition (50
). Furthermore, the human class IA PI3K adapter subunit p85 was cloned by use of a technique that screened for target proteins of receptor tyrosine kinases using the phosphorylated carboxy-terminal tail of the EGFR as bait (49
). However, in other cells, including A431 and A549 cells, this association is not seen (51
). Since EGFR lacks the Yxx
M consensus motif recognized by the SH2 domains of the class IA PI3K adapters and ErbB-3 contains seven copies, PI3K signalling was proposed to occur through a heterodimerized EGFR–ErbB-3 complex (44
). The validity of this model depends upon the coordinated expression of EGFR and ErbB-3 in addition to the specific heterodimerization of these two receptor chains over other combinations. However, it has been reported that ErbB-2 preferentially heterodimerizes with EGFR, ErbB-3, and ErbB-4 (26
). Furthermore, some cells, such as PC12 and A549, do not possess ErbB-3 yet activate PI3K following EGF stimulation. It has been suggested that the adapter protein p120cbl
may mediate an interaction between the activated EGFR and the class IA p85-p110 PI3K heterodimer (50
). Other groups have reported that the class IA PI3K adapter p85 appears to be poorly recruited to EGFR-containing phosphotyrosine complexes (Fig. ). Consequently, in certain cell types, the EGF-stimulated increase in 3′ phosphorylated lipids may more accurately represent the activation of class II PI3K enzymes than a class IA p85-p110 heterodimer.
The identification of either EGFR or ErbB-2 in human tumors correlates with a poor prognosis (20
). A common alteration of the EGFR in human disease is the deletion of exons 2 to 7. Expression of the truncated receptor, termed EGFRvIII, confers a selective advantage in vivo, resulting in a transformed phenotype. These cells also have been found to have high levels of constitutive PI3K activity (37
). This finding illustrates the need to improve the understanding of how EGFR regulates PI3K activity. Since EGF stimulation recruits both PI3K-C2α and PI3K-C2β to the EGFR-containing signaling complex, together with ErbB-2 (Fig. ), various SH2 domain-containing adapter proteins were examined to exclude any possible interaction with PI3K-C2β in vivo. Under conditions where PI3K-C2β was coimmunoprecipitated with the activated EGFR, there was no coimmunoprecipitation of this enzyme with molecules known to interact with the EGFR, namely p85α (23
), c-Src (30
), Shc (42
), and Grb2 (7
) (data not shown).
We demonstrate that the association of PI3K-C2β with EGFR is largely mediated by residue (p)Y992 but that (p)Y1068 and (p)Y1173 are also involved. These phosphotyrosine residues lie within the consensus sequence E(p)YL/I, which is also found on both the α and the β chains of the PDGFR at (p)Y579. Previously, this site on the PDGFR had been shown to bind only members of the Src family tyrosine kinases (36
). Such specificity contrasts with the behavior of class I PI3K adapters, whose SH2 domains preferentially bind (p)Yxx
M motifs. Furthermore, it supports our finding that class IA PI3K adapters do not mediate the recruitment of class II PI3K enzymes to growth factor receptors.
The N-terminal region (residues 1 to 301) of PI3K-C2β is able to associate with the EGFR (Fig. ). Unfortunately, the mechanism responsible for this interaction is currently unclear, although work is currently in progress to define the nature of this interaction. The fact that this domain associates weakly with the nonphosphorylated receptor may explain our findings that PI3K-C2β is observed in complex with the EGFR in quiescent cells.
It is currently unclear why receptor tyrosine kinases would need to recruit two distinct forms of the PI3K enzyme. While each PI3K enzyme may fulfill a specific biological role, the class I catalytic subunits utilize a broader range of phospholipid substrates in vitro than the class II enzymes. Consequently, stimulation of a class I PI3K enzyme alone could explain the activation of downstream targets, such as p70S6
), PDK1 and Akt (1
), and noncanonical isoforms of protein kinase C (38
). Although the kinetics with which the p85-p110 heterodimer and PI3K-C2α associate with the EGFR suggest temporal specificity, the same is not true for PI3K-C2β. Several ligands have now been demonstrated to mediate the activity of class II PI3K enzymes (5
). However, receptor heterogeneity makes it difficult to conclude how this activation is achieved. The results presented here also contrast with those obtained with other ligands, since neither EGF nor PDGF stimulated an increase in the total pool of either PI3K-C2α or PI3K-C2β lipid kinase activity (Fig. and data not shown). This fact may reflect a difference in receptor regulation but perhaps more likely indicates that both growth factors activate only a small proportion of the total pool of enzyme, as previously described for the class IA heterodimers (13
). In other cell types, where a larger number of PI3K molecules become activated, higher specific activity of immunoprecipitated PI3K is achieved.
It was previously shown that the lipid kinase activity of PI3K-C2β could be distinguished from those of other PI3K enzymes on the basis of its cation preference (2
). We have now expanded this work and demonstrated that in contrast to the class IA PI3K and PI4K enzymes (16
), PI3K-C2α and PI3K-C2β both can use Ca2+
as a cofactor for phosphate transfer (Fig. ). While p110α and PI3K-C2β can phosphorylate PtdIns in the presence of Mn2+
, PI3K-C2α cannot. Also, when PtdIns(4)P was used as a substrate, this cation selectivity was no longer observed, and all three enzymes were active only in the presence of Mg2+
. How these findings relate to the activity of each enzyme in situ remains uncertain, but this property could be used in future studies to characterize an isolated PI3K enzyme prior to its specific analysis. More importantly, perhaps, the data elegantly show that despite a high degree of sequence homology within the catalytic domains of these molecules, functional differences do exist. Structure-function studies have already begun to characterize the catalytic domains of PI3K enzymes in detail (63
) and will doubtless continue until their structures are solved. Ultimately, such differences might be exploited to develop selective antagonists against these enzymes to provide possible therapeutic benefits.
A long-overlooked property of the class IA enzymes is their serine-threonine protein kinase activity. It has been proposed that this activity provides negative feedback regulation, since serine phosphorylation of p85 results in decreased lipid kinase activity of the associated catalytic subunit (12
). Potentially, these enzymes could mediate downstream signaling via this protein kinase activity, but aside from IRS-1, no other substrates have been identified. However, in our experiments, neither PI3K-C2α nor PI3K-C2β displayed any protein kinase activity (data not shown), and although it was claimed in an earlier study (34
) that PI3K-C2γ can act as a protein kinase, no data were presented in that report. It remains to be seen if the differential regulation of protein kinase activity can provide the required functional specificity. Study of the class IB PI3K p110γ does indicate, however, that its lipid and protein kinase activities may regulate two distinct downstream pathways via Akt and mitogen-activated protein kinase, respectively (4
). If the class IA and class II PI3K enzymes do have different downstream targets, the mechanisms of their selective activation will require elucidation.