The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C.
Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)
Clawson
G A
GA
Department of Pathology, George Washington University, Washington, DC 20037.
Wang
Y F
YF
Schwartz
A M
AM
Hatem
C L
CL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000494:pharmacology; D034904:Lamin Type A; D034882:Lamins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000378:metabolism*; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D010446:Peptide Fragments / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000276:immunology / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C440308:lamin C; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D010450:Endopeptidases; D043583:Nucleoside-Triphosphatase