Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.
The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.
Otto
H
H
Freie Universität Berlin, Institut für Chemie-Biochemie, Berlin, Germany.
Dreger
M
M
Bengtsson
L
L
Hucho
F
F
D000818:Animals; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D010749:Phosphoprotein Phosphatases