Subcellular distribution of the TSC2 gene product tuberin in human airway smooth muscle cells is driven by multiple localization sequences and is cell-cycle dependent

The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin (TSC1 and 2), form a heteromer, which represses the kinase mammalian target of rapamycin. Loss of TSC1 or 2 results in diseases characterized by loss of cell-cycle control, including TSC and lymphangioleiomyomatosis. As tuberin has multiple signaling inputs, including phosphatidylinositide-3-OH kinase, mitogen-activated protein kinase, and adenosine monophosphate kinase, we postulated tuberin would have multiple protein interactions governed by subcellular localization and cellular status and examined this in primary human airway smooth muscle cells. Using immunofluorescence and confocal microscopy, tuberin was detected in cytoplasm, nucleus, nucleoli, and mitochondria. Fractionation of synchronized airway smooth cells showed that tuberin enters the nucleus in late G1, and passage through the cell cycle is necessary for nuclear entry. Deletion constructs showed localization sequences for the nucleus between amino acids 1351 and 1807, for mitochondria between 901 and 1350, and for cytoplasmic speckles between 1 and 450. Using fluorophore-tagged proteins, we observed fluorescence resonance energy transfer between tuberin and hamartin within these speckles, indicating a direct interaction between the proteins at this site. The observations that tuberin is localized to mitochondria and translocated to the nucleus in G1 are novel and consistent with interactions with proteins within multiple signaling pathways. Dynamic relocalization of tuberin may control these interactions to integrate these pathways. As tuberin has potential roles in proliferation, migration, and cell phenotype, it therefore warrants further investigation in diseases categorized by abnormalities in airway smooth muscle.