Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of t he inner nuclear membrane, appears to be involved in the spatial organizati on of the interface between nucleoplasma, lamina; and nuclear envelope. Its ability to interact with other proteins and the structural integrity of th e nuclear envelope is probably regulated by phosphorylation. Here, we repor t nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in th e native protein when purified from nuclear envelopes of mouse neuroblastom a Neuro2a cells. Five phosphorylation sites were detected by nano-electrosp ray mass spectrometric analysis of tryptic LAP 2 beta peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing o f these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a r egion known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases,Ser 179 is part of a consensus site for protei n kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Addition ally, we identified for the first time at the protein level the LAP 2 splic e variant LAP 2 epsilon in nuclear envelopes.