FIGURE 6. HGF hydrolyzes nuclear PIP₂.

A, lipid samples were spotted onto nitrocellulose membranes (right), as illustrated by the strip template (left), and the PIP₂ levels were detected using an anti-PIP₂ monoclonal antibody that reacts with PIP₂ with a high degree of specificity. Left column, experimental samples; middle column, PIP₂ standards; right column, PIP controls (20 pmol each). PI(3)P, phosphatidylinositol-3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; and PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate. B, densitometric measurement shows that AVP hydrolyzes 22 ± 2% of PIP₂ in whole cell preparations (n = 3, *, p < 0.05), whereas HGF stimulation does not hydrolyze significant amounts of PIP₂ in such preparations. However, upon HGF stimulation, PIP₂ levels in the nucleus are decreased by 87 ± 13% (n = 3, *, p < 0.05). Total lipids and nuclear lipids were isolated 4 min after stimulation with HGF (100 ng/ml) or 30 s after stimulation with AVP (100 nm). Values mean ± S.E. C, phospholipase C-γ1 is found in both the cytoplasm and the nucleus of SkHep1 cells. Non-Nuc., non-nuclear.