SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles*

Abstract

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P₃ phosphatases. Therefore, we investigated the dephosphorylation of [³²P]PtdIns(3,4,5)P₃ specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P₃ phosphatase assays revealed the production of both [³²P]PtdIns(3,4)P₂ and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P₃ 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P₃ 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [³²P]PtdIns(3,4,5)P₃ dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P₃ into PtdIns(3,4)P₂ within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P₃ phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.