Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope

The nuclear envelope separates the nucleoplasm from the rest of the cell. T hroughout the cell cycle, its structural integrity is controlled by reversi ble protein phosphorylation. Whereas its phosphorylation-dependent disassem bly during mitosis is well characterized, little is known about phosphoryla tion events at this structure during interphase. The few characterized exam ples cover protein phosphorylation at serine and threonine residues, but no t tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation oc cur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Ty rosine kinase and phosphatase activities remain associated with purified nu clear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envel ope proteins suggests that the same tyrosine kinases act at the nuclear env elope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope prot eins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antib odies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inne r nuclear membrane protein LAP2 beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.