Henning Otto, Phone: +49-30-838 56425, eMail: firstname.lastname@example.org
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. Our aims are to characterize the protein composition of the inner nuclear membrane, to identify phosphoproteins of the nuclear envelope and their binding partners, and to define their phosphorylation-dependent interactions.
Identification of Nuclear Envelope Proteins
The nuclear envelope consists of an inner and an outer nuclear membrane that are interconnected at the nuclear pore complexes. The outer nuclear membrane is continuous with the membrane of the endoplasmic reticulum. The exchange of macromolecules between cytoplasm and nucleus is tightly controlled by the nuclear pore complexes. This applies also for the translocation of membrane proteins to the inner nuclear membrane. Up to now, most of the few identified proteins of this membrane like, for example, the lamin B receptor bind with high affinity to the lamins which themselves form a very stable meshwork of intermediary filaments underlining the inner nuclear membrane. Other binding partners of such proteins are components of the chromatin. Both types of interactions confer to these inner nuclear membrane proteins a role in the spatial organization of the cell nucleus.
Since it is difficult to separate nuclear envelope substructures, little
is known about proteins specifically located to the inner nuclear membrane.
Therefore, we try to identify novel proteins at this membrane by subcellular
fractionation, two-dimensional gel electrophoresis and proteinchemical
methods, using mass spectrometric techniques and database searches
for the final protein identification. In this context, we are particularly
interested in refining methods to separate and handle membrane proteins.
Phosphorylation at the Nuclear Envelope
The nuclear envelope disassembles during mitosis. This is mainly driven by the mobilization of lamins and nuclear membrane proteins by cyclin-dependent protein kinases and other kinases, for example protein kinase C, at mitotic sites and is reverted at the end of mitosis by dephosphorylation of such sites. While mitotic phosphorylation is well characterized, little is known about protein phosphorylation during the other phases of the cell cycle.
We study phosphorylation at the nuclear envelope using intact cells
and isolated nuclear envelopes. In particular, we examine tyrosine phosphorylation
at the nuclear envelope. We started to identify several tyrosine-phosphorylated
in nuclear envelope preparations from Neuro2a mouse neuroblastoma cells.
Besides identifying phosphoproteins of the nuclear envelope and phosphorylation
sites within these proteins, we will also try to identify protein kinases
and phosphatases active at the nuclear envelope in order to characterize
possible signalling events at this structure.
Protein-Protein Interactions at the Nuclear Envelope
Finally, we search for binding partners for proteins of the inner nuclear membrane. We expect to get hints towards the functions of such proteins. At present, we focus on the lamina-associated polypeptide 2 beta (LAP 2 beta) which is an integral inner nuclear membrane protein and binds to chromatin and to lamins. LAP 2 beta belongs to a family of five isoforms, four of which contain transmembrane domains at the carboxy terminus. We identified two of these isoforms, LAP 2 beta and LAP 2 epsilon, in nuclear membrane preparations at the protein level.
We use LAP 2 beta as a model protein for the characterization of phosphorylation-dependent protein-protein interactions at the inner nuclear membrane. LAP 2 beta phosphorylation at mitotic sites is part of the mechanism by which the nuclear envelope disassembles during mitosis. LAP 2 beta is also phosphorylated during interphase since we could identify four serine and threonine residues that are phosphorylated in endogenous LAP 2 beta prepared from isolated nuclear envelopes. Three of these residues form a highly phosphorylated domain in LAP 2 beta which may integrate signalling by multiple protein kinases (Dreger et al., 1999).
Next steps will be to affinity purify binding partners by using recombinant
LAP 2 beta and to isolate protein complexes that contain LAP 2 beta. Phosphorylation-dependent
interactions will be addressed by using suitable point mutants of LAP 2
beta. Finally, we will check the consequences of expressing epitope-tagged
forms or fusion proteins with GFP of different LAP 2 beta mutants in living
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