Mitotic Phosphorylation of the Lamin B Receptor by a Serine/Arginine Kinase and p34cdc2
Journal of Biological Chemistry
The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that is modified at interphase by a nuclear envelope-bound protein kinase. This enzyme (RS kinase) specifically phosphorylates arginine-serine dipeptide motifs located at the NH 2 -terminal domain of LBR and regulates its interactions with other nuclear envelope proteins. To compare the phosphorylation state of LBR during interphase and mitosis, we performed phosphopeptide mapping of in vitro and in vivo 32
... in vivo 32 P-labeled LBR and analyzed a series of recombinant proteins and synthetic peptides. Our results show that LBR undergoes two types of mitotic phosphorylation mediated by the RS and the p34 cdc2 protein kinases, respectively. The RS kinase modifies similar sites at interphase and mitosis (i.e. Ser 76 , Ser 78 , Ser 80 , Ser 82 , Ser 84 ), whereas p34 cdc2 mainly phosphorylates Ser 71 . These findings clarify the phosphorylation state of LBR during the cell cycle and provide new information for understanding the mechanisms responsible for nuclear envelope assembly and disassembly. The nuclear lamina is a filamentous meshwork underlying the inner nuclear membrane (1, 2). In most cells this structure is a heteropolymer of type A and B lamins (3) linked to the inner nuclear membrane through integral membrane proteins. These lamin-binding proteins include the lamin B receptor (LBR 1 or p58; Ref. 4) and the lamina-associated polypeptides (5). LBR possesses a long, hydrophilic NH 2 -terminal domain protruding into the nucleoplasm, eight hydrophobic segments that are predicted to span the membrane, and a hydrophilic COOHterminal domain (6, 7). The NH 2 -terminal domain of LBR contains distinct sites for protein kinase A and p34 cdc2 kinase phosphorylation (8, 9) as well as a stretch rich in arginine-serine (RS) motifs (10). The RS motifs are specifically modified by a protein kinase that co-isolates with LBR and is part of a multimeric complex (8, 10). This LBR complex also includes the nuclear lamins and three polypeptides with molecular masses of 18 (p18), 150 (p150), and 34 (p34/p32) kDa, respectively (for pertinent information see Refs. 8, 10, and 12) . The latter protein has been shown to interact with the splicing factor 2 (SF2) as well as with the HIV-1 proteins . Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex (11). At the onset of mitosis, the structure of the nuclear envelope is dramatically altered. The nuclear lamina depolymerizes as a result of hyperphosphorylation of the nuclear lamins at specific sites involved in lamin-lamin (16), lamin-chromatin (17), and lamin-membrane (5) interactions. Following depolymerization, the bulk of type A lamins disperse in the cytoplasm, whereas type B lamins remain bound to remnants of the nuclear envelope. At the same time, the nuclear envelope membranes break down into vesicular structures (1). Apart from lamin hyperphosphorylation, Courvalin et al. (9) also reported that LBR is phosphorylated on serine and threonine residues during mitosis. As the events responsible for nuclear membrane breakdown are not completely understood and in light of the fact that LBR is phosphorylated by the RS kinase during interphase, we found it important to examine the specific modifications of LBR during mitosis. Results presented below reveal that during mitosis LBR is phosphorylated by both RS and p34 cdc2 protein kinases.