Interphase phosphorylation of the Drosophila nuclear lamin: site-mapping using a monoclonal antibody.
The Drosophila nuclear lamin is highly phosphorylated during interphase. Two interphase isoforms, differing in degree of phosphorylation, can be distinguished by one-dimensional SDS-polyacrylamide gel electrophoresis. One migrates with an apparent mass of 74 kDa (lamin Dm1); the other is more highly phosphorylated and migrates as a 76 kDa protein (lamin Dm2). We generated a monoclonal antibody, ADL84 which binds to lamin Dm1 but not lamin Dm2. Binding of ADL84 to lamin Dm2 was restored by phosphatase treatment of immunoblots containing lamins. Immunoprecipitation with ADL84 demonstrated that purified Drosophila nuclear lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. Indirect immunofluorescence experiments suggest that lamin Dm1 is present in all Drosophila cell types. The epitope for ADL84 was mapped by analyzing binding to bacterially expressed lamin deletion mutants and subsequently by screening for point mutants (randomly generated by polymerase chain reaction) which were not recognized by ADL84. The ADL84-epitope encompasses amino acids R22PPSAGP (arginine 22-proline 28). Peptide competition experiments demonstrated directly that phosphorylation of serine 25 impedes lamin binding by ADL84. This suggests that serine 25 is the lamin Dm2-specific phosphorylation site.
Department of Pharmacological Sciences, University Medical Center, SUNY at Stony Brook 11794-8651, USA.
N
N
Stuurman
N
N
Maus
P A
PA
Fisher
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001483:Base Sequence; D001667:Binding, Competitive; D004330:Drosophila / Q000378:metabolism*; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D007399:Interphase / Q000502:physiology*; D034882:Lamins; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry
D000911:Antibodies, Monoclonal; D034882:Lamins; D009687:Nuclear Proteins; D012694:Serine