Expression of Chicken Lamin B2 in Escherichia coli: Characterization of its Structure, Assembly, and Molecular Interactions E. Heitlinger,* M. Peter,* M. H/iner,* A. Lustig,§ U. Aebi,*ll and E. A. Nigg* * M. E. Mtiller-Institute at the Biocenter, University of Basel, CH-4056 Basel, Switzerland; ~Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland; §Department of Biophysical Chemistry, Biocenter, University of Basel; II Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Abstract. Chicken lamin B2, a nuclear member of the dimers is in striking contrast to the lateral mode of as- Downloaded from http://rupress.org/jcb/article-pdf/113/3/485/1061190/485.pdf by guest on 25 September 2021 intermediate-type filament (IF) protein family, was ex- sociation observed previously for cytoplasmic IF pressed as a full-length protein in Escherichia coli. dimers. At the third level of organization, these polar After purification, its structure and assembly proper- head-to-tail polymers further associated laterally, in an ties were explored by EM, using both glycerol spray- approximately half-staggered fashion, to form filamen- ing/low-angle rotary metal shadowing and negative tous and eventually paracrystal-like structures reveal- staining for preparation, as well as by analytical ing a pronounced 24.5-nm axial repeat. Finally, fol- ultracentrifugation. At its first level of structural orga- lowing up on recent studies implicating the mitotic nization, lamin B2 formed "myosin-like" 3.1S dimers cdc2 kinase in the control of lamin polymerization consisting of a 52-nm-long tail flanked at one end by (Peter, M., J. Nakagawa, M. Dor6e, J. C. Labb6, and two globular heads. These myosin-like molecules are E. A. Nigg. 1990. Cell. 61:591-602), we have exam- interpreted to represent two lamin polypeptides inter- ined the effect of phosphorylation by purified cdc2 acting via their 45-kD central rod domains to form a kinase on the assembly properties and molecular inter- segmented, parallel and unstaggered 52-nm-long two- actions of the bacterially expressed lamin B2. Phos- stranded u-helical coiled-coil, and their COOH- phorylation of chicken lamin B2 by cdc2 kinase inter- terminal end domains folding into globular heads. At feres with the head-to-tail polymerization of the lamin the second level of organization, lamin BE dimers as- dimers. This finding supports the notion that cdc2 ki- sociated longitudinally to form polar head-to-tail poly- nase plays a major, direct role in triggering mitotic mers. This longitudinal mode of association of lamin disassembly of the nuclear lamina. AMINS, having molecular masses between 60 and 75 kD, terminus, and probably arises from alternate RNA splicing are member~ of the intermediate-type filament (IF) ~ (McKeon et al., 1986; Fisher et al., 1986; Riedel and Wer- protein family (McKeon et al., 1986; Fisher et al., ner, 1989). In birds, three structurally distinct lamin iso- 1986; Franke, 1987). In situ, they form a karyoskeletal types have been identified; according to the homologies of structure, known as the nuclear lamina. This fibrillar mesh- their amino acid sequences (Vorburger et al., 1989a; Peter work lines the inner nuclear membrane (Gerace et al., 1978; et al., 1989) and their protein chemical properties (Lelmer Krohne and Benavente, 1986; Aebi et al., 1986; Gerace and et al., 1986; Stick et al., 1988), they have been designated Burke, 1988; Nigg, 1989; Paddy et al.; 1990) and is thought as lamins A, B1, and 132. Whereas lamin 132 is the quantita- to interact with interphase chromatin (Gerace et al., 1978; tively predominant B-type lamin in chicken, it represents a Hancock, 1982; Lebkowski and Laemmli, 1982; Paddy et minor component in most mammalian tissues thus far inves- al., 1990; Glass and Gerace, 1990). In mammals, lamins tigated; conversely, lamin B1 is a minor component in most have been classified as either A-, or B-type, based on protein avian tissues, but it is closely related to the predominant chemical and primary sequence criteria (see Gerace and B-type lamin in mammals (Lehner et al., 1986). The exis- Burke, 1988; H6ger et al., 1988; Peter et al., 1989; Nigg, tence of BI- and B2-type lamin polypeptides in both mam- 1989). In addition, mammals express a C-type lamin, but mals and amphibians has recently been confirmed by protein this protein is identical to lamin A except for its COOH sequence analysis of purified murine lamin B (Weber et al., 1990), and by cDNA cloning of murine and Xenopus laevis 1. Abbreviations used in this paper: IP~, isopropyl-/~-D-thiogalactopyrano- lamin B2 (H6ger et al., 1990). side; MES, 2-morpholino-ethanesulfordc acid. Lamins exhibit structural features common to all IFs (Aebi © The Rockefeller University Press, 0021-9525/91/05/485/11 $2.00 The Journal of Cell Biology, Volume 113, Number 3, May 1991 485-495 485 et al., 1986, 1988; Parry et al., 1986, 1987; Stewart, 1990; Stewart et al., 1989; Hatzfeld and Weber, 1990; Coulombe Moir et al., 1990). Specifically, they contain a conserved rod and Fuchs, 1990) or nuclear lamins (Ward and Kirschner, domain consisting of four a-helical coils (i.e., 1A, 1B, 2A, 1990; Moir et al., 1990) in bacteria offers a powerful tool 2B) connected by linkers and flanked by end domains of vari- to systematically investigate the structure, assembly, and able size and sequence (McKeon et al., 1986; Fisher et al., molecular interactions of both wild-type and mutated IF 1986; Parry et al., 1986). Compared with vertebrate cyto- polypeptides in vitro. plasmic IFs, coil 1B in lamins has a 42-amino acid exten- Here we describe the expression of full-length chicken sion, yielding a 45- instead of a 40-kD rod domain. From lamin B2 in Escherichia coil using an expression plasmid an evolutionary point of view, it is intriguing that an identi- containing the T7 promoter (Rosenberg et al., 1987). The cally increased rod length is observed also in cytoplasmic IFs purified recombinant protein is characterized in terms of its of invertebrates (Weber et al., 1988, 1989). At their first in vitro structure, assembly, and molecular interactions. We level of structural organization, two lamin polypeptides as- have also performed preliminary experiments exploring the sociate via their rod domains into parallel, unstaggered two- effect of phosphorylation by the cdc2 kinase on the molecular stranded o~-helical coiled-coil dimers (see Parry et al., 1986; interactions of this bacterially expressed chicken lamin B2. Krohne et al., 1987). In the electron microscope lamin dimers resemble "myosin-like" molecules in that they reveal a 50-53 nm long tail segment flanked at one end by two Materials and Methods globular "headsY (We note that the terms "head" and "tail" are occasionally used to designate NH2 and COOH termini Construction of a Chicken Lamin Be when describing the tripartite organization of IF proteins. Expression Plasmid Downloaded from http://rupress.org/jcb/article-pdf/113/3/485/1061190/485.pdf by guest on 25 September 2021 These designations should not be confounded with the mor- The chicken lamin 132 eDNA (Vorburger et al., 1989a) was cloned into the phological descriptions used here. In particular, we empha- expression vector pAR3038 (Rosenberg et al., 1987), which allows selec- size that the "head" structures discussed here, i.e., those that tive expression of the cloned eDNA under the control of the T7 promoter. To express lamin Be from its proper ATG start codon, an NdeI restriction can be seen by EM, are formed by the COOH-terminal end site was introduced into the 5' end of the lamin B2 eDNA (Fig. 1). For this domains of lamins.) (Aebi et al., 1986, 1988; Moir et al., 1990). Under appropriate in vitro conditions, lamin dimers assemble into IF-like structures, and ultimately into para- NdeI crystalline arrays with a characteristic 24.5-nm axial repeat (Goldman et al., 1986; Aebi et al., 1986; Parry et al., 1987; 5' - GCGAGCTGAGAATTCA~CAGGCA - 3' Moir et al., 1990). Barn}If During mitosis, lamins depolymerize and repolymerize in E~ EcoRI a time frame closely corresponding to disassembly and I l reformation of the nuclear envelope (Gerace et al., 1978; Gg Lamin B2 eDNA Krohne et al., 1978; Gerace and Blobel, 1980). Whereas I Ndel A-type lamins become completely solubilized upon disassem- NdeI t EcoRI EcoRI bly, B-type lamins remain associated with membranous I I structures (Gerace and Blobel, 1980; Stick et al., 1988; see also Burke and Gerace, 1986). Subcellular fractionation studies have revealed that after lamina depolymerization, lamins A and C in mammalian cells are dissociated into dimers or tetramers (Gerace and Blobel, 1980; Dessev et al., 1990). Disassembly of the nuclear lamina has long been ~Llgase proposed to be the consequence of hyperphosphorylation of 3) T4 - DNA - Ligase the lamin polypeptides by cell cycle-specific protein kinases t (Gerace and Blobel, 1980; Gerace et al., 1984; Miake-Lye and Kirschner, 1985; Ottaviano and Gerace, 1985; Dessev COOH- [ .i" NH2 et al., 1988). Strong support for this notion stems from the Gg Lamin B2 / finding that cdc2 kinase, a highly conserved regulator of the cell cycle (Nurse, 1990), phosphorylates lamins at M phase- specific sites (Peter et al., 1990; see also Ward and Kirsch- ner, 1990), and, concomitantly, is able to depolymerize the nuclear lamina upon incubation with nuclei (Peter et al., 1990). Moreover, mutations affecting mitotic phosphoryla- tion sites have been shown to interfere with lamina disassem- bly in vivo (He.aid and McKeon, 1990). Site-specific phos- phorylation has been shown to induce disassembly, or inhibit Figure 1. Construction of a chicken lamin B~ baeteriN expression plasmid. For insertion of the eDNA into the NdeI site of the expres- assembly, of cytoplasmic IFs both in vitro (see Inagaki et al., sion vector pAR3038 (Rosenberg et al., 1987), oligonueleotide- 1987; Geisler and Weber, 1988; Kitamura et al., 1989; Hisa- directed site-specific mutagenesis was used to create an NdeI site naga et al., 1990; Chou et al., 1990) and in mitotic cells (see within the translational start codon ATG of the lamin B2 eDNA.