Eukaryotic cells widely use the phosphorylation of proteins to transmit and integrate signals received from their environment and to regulate cellular functions. In the context of signal transmission towards the cell nucleus, we are studying the role of the inner nuclear membrane of the nuclear envelope in signal transduction and regulation of nuclear function. In particular, we are interested in protein phosphorylation at this structure and the functional role of such phosphorylation. We aimed to identify phosphoproteins of the nuclear envelope and to define their phosphorylation-dependent interactions.

Phosphorylation of LAP2-beta

Within the context of nuclear envelope signalling, we analyzed the interphase phosphorylation of the INM protein LAP-2b. In collaboration with Dr. G. Neubauer from Dr. M. Mann’s lab (EMBL, Heidelberg), we identified three phosphorylated serine / threonine residues of this protein (Dreger et al. (1999).

To address the functions associated with LAP-2b phosphorylation, their consequence for LAP2-beta localization, for its interactions with known interactors and for the structural and functional organization of the nucleus and for cellular functions like replication or transcription, we prepared point-mutated LAP2-beta mutants, in which the relevant serine / threonine residues are replaced by either aspartate or alanine residues.

Tyrosine phosphorylation at the nuclear envelope

More excitingly, we found reversible tyrosine phosphorylation at the nuclear envelope. We demonstrated tyrosine kinase and phosphotyrosine phosphatase activity at the nuclear envelope in vitro as well as in intact cells. This served as a starting point for studying tyrosine phosphorylation at the nuclear envelope. Besides other proteins, we identified LAP2-beta as an INM protein correlated to a phosphotyrosine immunosignal (Otto et al. (2001)).

At that time, we were technically not able not identify a single tyrosine-phosphorylation sites of one of the identified proteins. In collaboration with Dr. Andreas Schlosser (Charité, Institut für Medizinische Immunologie), we now addressed again the tyrosine phosphorylation of nuclear-envelope proteins. Dr. Schlosser developed an extremely sensitive mass spectrometric method for identifying and analyzing phosphorylated, particularly tyrosine-phosphorylated peptides. This method enabled us to identify for the first time, besides some new serine / threonine phosphorylation sites, three tyrosine phosphorylation sites of the INM protein emerin. Emerin had not been found with our previous approach, since it had been outside the molecular weight range of the electrophoretic system used. The identification of further emerin tyrosine-phosphorylation sites by Dr. Ramars Amanchy (McKusick-Nathans Institute for Genetic Medicine and Dept. of Biological Chemistry, Johns Hopkins University) confirmed and complemented our own data (Schlosser et al. (2006)).

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Henning Otto / updated: 12.04.2011