We examined the structural features that determine p110β nuclear localization. Whereas overexpressed recombinant p110β remains mainly cytoplasmic, transfected rp110β in combination with rp85β, but not rp85α, localizes to the nucleus. Although the ubiquitous catalytic and regulatory subunits form all possible heterodimeric combinations (16
), only p85β/p110β complexes localize efficiently in the nucleus. The search for nuclear localization motifs in p85β and p110β yielded several candidate sequences, but only mutation of the NLS located within the p110β C2 domain significantly reduced p110β nuclear localization. The fact that p110β alone does not enter the nucleus suggests that p110β must associate with p85β for its NLS motif to be functional; the predicted quaternary structure of p85β/p110β reported here supports this possibility (see below). p110β nuclear PI3K activity is maximal in S phase (35
), suggesting that p85β/p110β complexes shuttle in and out of the nucleus. We identify here a functional NES in p85β which, when deleted, increases p85β/p110β nuclear localization.
Proteins enter the nucleus through nuclear pores, large macromolecular complexes composed of nucleoporins. Understanding of macromolecular transport processes across the nuclear envelope has increased in recent years, and many transport receptors have been identified. Most of these receptors are similar to the import receptor importin β (karyopherin β). Members of this family have been classified as importins or exportins, and both types are regulated by the GTPase Ran. Importins recognize their substrates in cytoplasm and transport them to the nucleus; once in the nucleus, RanGTP binds to importins, inducing the release of import cargoes. In contrast, exportins interact with their substrates only in the nucleus in the presence of RanGTP and release them after GTP hydrolysis in the cytoplasm (reviewed in reference 49
). Nuclear import and export are multistep processes initiated by the recognition of NLSs or NESs. The most thoroughly examined import signal (“classical” and bipartite NLS) contains multiple basic residues. Their transport is mediated by importin β, which directly associates these NLS via the adaptor protein importin α (49
). The functional NLS in p110β is homologous to that found in class II PI3KC2α, which also transits to the nucleus (10
), suggesting potential conservation of structural features for nuclear import between PI3K classes.
The best-studied exportins are Crm1/Xpo1, which recognizes leucine-rich NES. Crm1 forms a stable ternary complex with Ran-GTP and with NES cargoes that can exit the nucleus. Studies of Crm1-mediated export were aided by the discovery of the antifungal agent leptomycin B, a highly specific and potent inhibitor of Crm1 function (11
). Of the three potential NES sequences in p85β, only the one located at the N terminus regulated the nuclear localization of p85β/p110β. In the p85β/p110β complex, p110β therefore contributes by providing the NLS, whereas p85β supplies a functional NES, showing that this complex acts as a single entity for nuclear transport. Indeed, the predicted structure of p85β/p110β described here (based on that in reference 33
) shows that the NLS sequence in the C2 domain is in close proximity to p85β, supporting the possibility that p110β association to p85β alters p110β structure in this region to yield a functional NLS.
Neither p110β nor p85β is exclusively nuclear; the cytoplasmic forms might represent complexes with p85α and p110α, respectively, or p85β/p110β complexes in transit from both compartments. In the case of p85β, its overexpression renders a fraction of this protein nuclear, suggesting that it associates with other NLS-containing proteins. In support of this possibility, p85α and, to a greater extent, p85β, associates with X-box binding protein 1 (XBP1), modulating the nuclear localization of this transcription factor (which contains an NLS) (46
). Similarly, in the case of p110β, association with p85β is critical for its translocation to the nucleus; however, other p110β-associated nuclear proteins (such as PCNA or Nbs1 [32
]) might facilitate the translocation of p110β to the nucleus in p85β-deficient cells.
We focused on a comparison of the class IA
PI3K isoforms p110α and p110β; there is nonetheless an additional class IA
isoform, p110δ, as well as the closely related class IB
p110γ isoform, which associates with p101 and p84 regulatory subunits (50
). When overexpressed in HepG2 cells, p110γ localizes to the nucleus after serum treatment; in this case, interference of p110γ association with p101 increases p110γ nuclear localization (41
). There is no region homologous to that of the p110β C2 domain in p110γ (45
), although we found polybasic motifs in the N terminus, in the helical domain, and at the beginning of the C2 domain (data not shown). Alignment of the NLS region in p110β and p110α () shows that most of this basic motif in p110β is lost in p110α. Comparison of the p85α/p110α structure (24
) to the p85β/p110β structural prediction described here () also shows that the loop in which p110β NLS localizes is much shorter in p110α. These observations might explain why a large proportion of p110β, but not of p110α, localizes to the nucleus. An in silico
search for p110β NLS homologues in p110δ, as well as p110δ structure (4
), showed a similar polybasic region in p110β and p110δ C2 domains; further study is needed to define whether the p110δ motif is a functional NLS.
The first report of nuclear PIP3
showed rapid translocation of a PIP3
-binding protein (PIP3
BP), which is abundant in brain, to the nuclei of the rat pheochromocytoma PC12 cell line after NGF treatment, as well as in PDGF-treated NIH 3T3 cells (51
). In human promyelocytic HL60 cells, both retinol and vitamin D3
induced differentiation to granulocytes or monocytes, respectively, and triggered an increase in nuclear p85 staining (reviewed in references 36
). In all of these cases, the authors defined the specific isoform localizing to the nucleus. The negative regulator of PI3K, PTEN, is also reported to transit to the nucleus and regulate cell survival (17
). In the case of nuclear PI3K in PC12 cells, a nucleus-specific phospholipase C activates a neuron-specific GTPase, PIKE (phosphoinositide 3-kinase enhancer), which is able to increase nuclear PI3K activity (56
). Isolated nuclei from PC12 cells treated with NGF or transfected with active PI3K were resistant to DNA fragmentation factor caspase-activated DNase (DFF40/CAD); interference with p110α diminished NGF protection from apoptosis, supporting p110α control of nuclear PIP3
in PC12 cells (1
The antiapoptotic function of nuclear PIP3
in PC12 cells is proposed to result from PIP3
binding to B23 nucleophosmin, a protein that inhibits DFF40/CAD (20
). Other authors have suggested that nuclear PKB function in NGF-treated PC12 cells is mediated by PKB phosphorylation of acinus, resulting in acinus inhibition of apoptotic chromatin condensation (23
). A third mechanism has been reported for the function of nuclear PI3K in NGF-treated PC12 cells, PCKζ-PI3K-dependent nuclear translocation, which mediates phosphorylation of nucleolin, a stabilizing agent for the antiapoptotic protein Bcl-2 (48
). These results indicate that in some cell types (PC12 cells), the neurotrophin NGF activates nuclear PI3K, which in turn induces cell survival.
We report here that the p85β/p110β complex localizes to the nucleus in several cell types. This translocation is regulated by an NLS sequence in the p110β C2 domain and by an NES in the p85β N-terminal domain. Our results demonstrate that the p85β/p110β complex regulates cell viability only when it is correctly localized at the cell nucleus.