Evidence of SHIP2 Ser¹³² phosphorylation, its nuclear localization and stability

PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ are major signalling molecules in mammalian cell biology. PtdIns(3,4)P₂ can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5)P₃ levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser¹³², Thr¹²⁵⁴ and Ser¹²⁵⁸; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser¹³² was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser¹³² demonstrated PtdIns(4,5)P₂ phosphatase activity. Endogenous phospho-SHIP2 (Ser¹³²) showed an overlap with PtdIns(4,5)P₂ staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5)P₃, and in the nucleus where it may interact with PtdIns(4,5)P₂, particularly in speckles.