Abstract
LAP2alpha is a LEM family protein associated with nucleoplasmic A-type lamins and chromatin in interphase. Like lamins and other lamina proteins LAP2alpha is cytoplasmic in metaphase, but it associates with chromosomes prior to nuclear envelope formation in late anaphase to telophase. In vitro phosphorylation analysis and mass spectrometry identified a cluster of at least three mitotic cyclin-dependent kinase 1 phosphorylation sites in the C-terminal chromatin-binding region of LAP2alpha as well as four additional potential sites in the cluster, some of which were targeted alternatively in LAP2alpha mutated at the major sites. LAP2alpha mutants containing serine --> alanine mutations at all seven sites revealed a clear phenotype. Mutated LAP2alpha remained associated with chromosomes throughout mitosis, but the dissociation of lamins into the cytoplasm and nuclear envelope disassembly were not affected. These data demonstrate the in vivo significance of mitotic phosphorylation for the dynamic behavior of LAP2alpha in the cell cycle and show that, unlike the interaction with lamins, the chromatin association of LAP2alpha is regulated by multiple mitosis-specific phosphorylation at sites clustered within a defined region in the C terminus of the protein.
Highlights
In higher eukaryotes, lamins [1] and lamin-binding proteins form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane [2, 3]
Mutated LAP2␣ remained associated with chromosomes throughout mitosis, but the dissociation of lamins into the cytoplasm and nuclear envelope disassembly were not affected. These data demonstrate the in vivo significance of mitotic phosphorylation for the dynamic behavior of LAP2␣ in the cell cycle and show that, unlike the interaction with lamins, the chromatin association of LAP2␣ is regulated by multiple mitosis-specific phosphorylation at sites clustered within a defined region in the C terminus of the protein
Isoelectric focusing revealed a more acidic pI of mitotic versus interphase LAP2␣ [27]. As these properties were consistent with a mitosis-specific phosphorylation of LAP2␣, we set out to test this possibility by various means
Summary
Lamins [1] and lamin-binding proteins form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane [2, 3]. These data demonstrate the in vivo significance of mitotic phosphorylation for the dynamic behavior of LAP2␣ in the cell cycle and show that, unlike the interaction with lamins, the chromatin association of LAP2␣ is regulated by multiple mitosis-specific phosphorylation at sites clustered within a defined region in the C terminus of the protein.
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