Mitotic Phosphorylation of the Lamin B Receptor by a Serine/Arginine Kinase and p34

Abstract

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 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 arginineserine (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 Rev and Tat (13-15). 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.

Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts

“…However, in the case of the RS protein, LBR, in chicken hepatoma cells, DU249, the level of phosphorylation was similar in I-phase and M-phase cells in vivo (13). In our cell-free system involving Xenopus egg cytosol fractions, the level of phosphorylation of LBR by SRPK was similar in S-phase and M-phase extracts (data not shown).…”

“…We demonstrated that the binding of LBR to chromatin is regulated by phosphorylation of LBR in the nucleoplasmic region by cell cycle-specific Xenopus egg cytosol fractions (6). It is also known that LBR is phosphorylated by cdc2 kinase (12,13), SR protein-specific kinase (SRPK) (14), protein kinase A (12), calmodulin-dependent protein kinase II (12), and casein kinase II (12). However, in the cases of all inner nuclear membrane proteins, the protein kinase responsible for the regulation of the binding to chromatin has not yet been identified.…”

“…Treatments were the following: control buffer (1), SC (2), SC containing 10 nM staurosporine (Sta.) (3), SC containing 1 mM A3 (4), SC containing 1 M K252b (5), SRPK1 with an ATP-regenerating system (6), SRPK1 without the ATP-regenerating system (7), SRPK1 with the ATP-regenerating system and 1 mM A3 (8), SRPK1 with the ATP-regenerating system and 1 M K252b (9), control buffer (10), SC (11), SRPK1 with the ATP-regenerating system (12), and SRPK1 without the ATP-regenerating system (13). Each column shows the mean Ϯ S.D.…”

Lamin B receptor

“…The role of phosphorylation in dismantling the NE and associated heterochromatin is not entirely understood; as exemplified by the LBR-lamin B-chromatin interaction, the binding may be weakened favoring dissociation. 17,56,57 However, in the studied case of chicken LBR, the CDK1 site(s) of mitotic phosphorylation are in dispute: one group 17 identified Thr 188 as the site and suggested that this might destabilize the LBR-lamin B interaction; the other group 57 identified Ser 71 and argued against a weakening of LBR-lamin B binding. More recent studies, 58,59 involving the binding of Xenopus sperm chromatin to LBR-GST beads, present evidence that during S phase the RS protein kinase stimulates LBR-chromatin binding by phosphorylation of one or more serine residues in the RS region (see Fig.…”

The Inner Nuclear Membrane Protein Lamin B Receptor Forms Distinct Microdomains and Links Epigenetically Marked Chromatin to the Nuclear Envelope

“…LBR is a polytopic inner nuclear membrane protein consisting of a long, N-terminal domain, seven or eight hydrophobic transmembrane regions, and a C-terminal tail (13). The N-terminal part of the molecule protrudes to the nucleoplasm and contains multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases (14,15); the hydrophobic region represents, instead, a (functional) form of sterol reductase and is involved in cholesterol metabolism (16).…”