Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4)

Abstract

Calcium (Ca²⁺) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca²⁺ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) within the nucleus, leading to Ca²⁺ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P₂ hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca²⁺ signaling and downstream events. We found that EGF-induced Ca²⁺ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca²⁺ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP₃) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P₂ by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP₃ modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Keywords: EGFR; calcium; calcium intracellular release; epidermal growth factor receptor (EGFR); inositol 1,4,5-trisphosphate (IP3); nucleus; phospholipase C; phospholipase Cδ-4; proliferation; protein kinase C (PKC).