Identification of phosphorylation sites on murine nuclear lamin C by RP‐HPLC and microsequencing

Abstract

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser ~9~' and Ser ~m, and possibly Ser 4°7 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser 39° and Ser ~: and to PKC at Ser 57-'. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cde2-kinase, PKC and PKA. The sequence data suggests that multiple mr~leeular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.

The lamin protein family

“…Lamins are phosphorylated by multiple kinases and contain many conserved phosphorylation sites, with more than 30 known sites in human A-type lamins alone [36]. Three kinases known to modify and modulate lamins' activities are CDC2, protein kinase C (PKC) and protein kinase A (PKA).…”

Partners and post-translational modifications of nuclear lamins

“…Lamin phosphorylation by CDK1 impedes assembly of head-to-tail polymers but does not disrupt lamin dimer formation (Heitlinger et al 1991; Peter et al 1991). CDK1 targets two regions important for head-to-tail association of lamin A dimers (Strelkov et al 2004); phosphorylation at Ser-22, and at Ser-392, Ser-404 and Ser-406 at the opposite end of the coiled-coil domain, are required to depolymerize lamin filaments during mitosis (Heald and McKeon 1990; Peter et al 1990; Ward and Kirschner 1990; Eggert et al 1991; Enoch et al 1991; Thompson and Fields 1996; Schneider et al 1999; Figure 2B). Phosphorylation of the A-type lamin in Drosophila , named lamin C, at Ser-37 (homologous to human lamins A/C Ser-22) increases the solubility of the lamin protein and eliminates its ability to interact with chromatin in vitro (Zaremba-Czogalla et al 2012).…”

Regulation of lamin properties and functions: does phosphorylation do it all?

“…In Xenopus , the predicted phosphoacceptor residues are S386/388, S391/393 and S395/397 for lamins A, B1 and B2, respectively. Phosphorylation of this region by Cdk1 was also confirmed experimentally for murine lamin A (S390/392) [ 25 , 26 ] and for chicken lamin B2 (S384/386) [ 34 ]. Near this Cdk1 site, there is a very conservative region among B1 lamins.…”

“…This may lead to a situation where a particular phosphoacceptor amino acid residue in a lamin may be phosphorylated by an entire set of kinases. The best-known example of this effect is the N-terminal 'mitotic' consensus site and nearby sites, which can be modified not only by Cdk1, but also by other Cdks, Gsk3, ERK1-2/ MAPK, PKA and PKC, and by virus kinases both in vitro and in vivo [23][24][25][26]31,32,35,54,55,[95][96][97][98]. This means that multiple sites of phosphorylation can be modified by a wide range of stimuli (i.e.…”