The last twenty residues in the head domain of mouse lamin A contain important structural elements for formation of head-to-tail polymers in vitro.
Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a alpha-helical rod domain flanked by non-alpha-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.
Isobe
Kazuhiro
K
Division of Biopolymer Research, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima, Saga, Japan.
Gohara
Rumi
R
Ueda
Toshihisa
T
Takasaki
Yozo
Y
Ando
Shoji
S
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D034904:Lamin Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid
D007381:Intermediate Filament Proteins; D034904:Lamin Type A; D011108:Polymers; D011994:Recombinant Proteins; D016203:CDC2 Protein Kinase