Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

Abstract

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2alpha and PI3K-C2beta act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2beta resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2beta co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2beta in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2beta into the nucleus.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

“…The unstimulated cells yielded a plasma membrane localization similar to that previously observed in SKBr3 breast cancer cells, suggesting constitutive activation of PI3K at the plasma membrane (Figure 3(a)). However, upon estrogen stimulation for 15 min, the reporter translocated to the nucleus, suggesting activation of PI3K in the nucleus, as has been suggested by studies characterizing a nuclear pool of PI3K [61, 62]. Importantly, the inactive stereoisomer of estrogen (17 α -estradiol) did not demonstrate PI3K activation, even at 1000x the concentration of 17 β -estradiol, demonstrating the stereoselectivity of the receptor involved for the physiologically active isomer of estrogen.…”

Regulation and cellular functions of class II phosphoinositide 3-kinases

“…A nuclear localization motif was observed in PI3K-C2α [39]. More recently, it has been reported that EGF stimulation increases levels of PI3K-C2β in the cytosol and in the nuclei where the enzyme appears to co-localize with lamin A/C [40]. An increase in nuclear PI3K-C2β levels and lipid kinase activity was reported upon EGF stimulation.…”

“…in the C-terminal C2 domain [40]. Whether translocation of the enzyme to the nucleus is related to activation of the enzyme remains to be established.…”

Nuclear PI3K signaling in cell growth and tumorigenesis

“…The nuclear production of PI(3,4,5)P 3 reflects the activities of various lipid kinases acting locally, and like PI(4,5)P 2 -binding proteins (Lewis et al, 2011 ), factors bound to PI(3,4,5)P 3 may spatially and temporally alter the activities of the lipid kinases and downstream signaling (Tanaka et al, 1999 ). Accumulating evidence supports the notion that nuclear lipid kinases either exhibit signal-dependent translocation from the cytoplasmic compartment or are native to the nucleus, where their focal distribution and activities are regulated by various signals (Neri et al, 1994 ; Banfic et al, 2009 ; Kumar et al, 2011 ). So far, class I and II PI3Ks and inositol polyphosphate multikinase (IPMK)/Ipk2 activities have been observed within the nucleus.…”