N. Stuurman et al., INTERPHASE PHOSPHORYLATION OF THE DROSOPHILA NUCLEAR LAMIN - SITE-MAPPING USING A MONOCLONAL-ANTIBODY, Journal of Cell Science, 108, 1995, pp. 3137-3144
The Drosophila nuclear lamin is highly phosphorylated during interphas
e. Two interphase isoforms, differing in degree of phosphorylation, ca
n be distinguished by one-dimensional SDS-polyacrylamide gel electroph
oresis. One migrates with an apparent mass of 74 kDa (lamin Dm(1)); th
e other is more highly phosphorylated and migrates as a 76 kDa protein
(lamin Dm(2)). We generated a monoclonal antibody, ADL84 which binds
to lamin Dm(1) but not lamin Dm(2). Binding of ADL84 to lamin Dm(2) wa
s restored by phosphatase treatment of immunoblots containing lamins.
Immunoprecipitation with ADL84 demonstrated that purified Drosophila n
uclear lamins Dm(1) and Dm(2) are present as a random mixture of homo-
and heterodimers. Indirect immunofluorescence experiments suggest tha
t lamin Dm(1) 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 (rand
omly generated by polymerase chain reaction) which were not recognized
by ADL84. The ADL84-epitope encompasses amino acids R(22)PPSAGP (argi
nine 22-proline 28). Peptide competition experiments demonstrated dire
ctly that phosphorylation of serine 25 impedes lamin binding by ADL84.
This suggests that serine 25 is the lamin Dm(2)-specific phosphorylat
ion site.