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

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2 alpha and PBK-C2 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-C2 beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2 beta 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-C2 beta 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-C2 beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2 beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent oil it 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 its all EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with ail efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2 beta in nuclei. Further, this effect is dependent oil a nuclear localization signal present within the C-terminal C2 domain. indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2 beta into the nucleus.