Abstract
The nuclear lamina can bind and sequester transcription factors (TFs), a function lost if the lamina is abnormal, with missing or mutant lamin proteins. We now show that TF sequestration is not all-or-nothing, but a dynamic physiological response to external signals. We show that the binding of the ubiquitous TF, Oct-1, to lamin B1 was reversed under conditions of cellular stress caused, inter alia, by the chemical methylating agent methylmethanesulfonate (MMS). A search for lamin B1 post-translational modifications that might mediate changes in Oct-1 binding using kinase inhibitors uncovered a role for c-Jun N-terminal kinase (JNK). Phosphoproteomic and site-directed mutagenesis analyses of lamin B1 isolated from control and MMS-treated nuclei identified T575 as a JNK site phosphorylated after stress. A new phospho-T575 specific anti-peptide antibody confirmed increased interphase cellular T575 phosphorylation after cell exposure to certain stress conditions, enabling us to conclude that lamin B1 acts as an interphase kinase target, releasing Oct-1 to execute a protective response to stress.
Highlights
The nuclear envelope (NE) is composed of the inner and outer nuclear membranes in addition to a nucleoplasmic meshwork of proteins, the nuclear lamins, which contribute to the structural integrity of the nucleus [1]
We have previously shown that Oct-1 sequestration at the NE is lost in the absence of full length lamin B1 in Lmnb1Δ/Δ mouse embryonic fibroblasts (MEFs)
The sequential extraction and Fluorescence loss in photobleaching (FLIP) experiments demonstrate that Oct-1 is less tightly associated within the nucleus upon MMS treatment they do not prove that this is due to a loss of direct interaction with lamin B1
Summary
The nuclear envelope (NE) is composed of the inner and outer nuclear membranes in addition to a nucleoplasmic meshwork of proteins, the nuclear lamins, which contribute to the structural integrity of the nucleus [1]. The NE is involved in various cellular processes including the organisation of chromosomes within the nucleus, DNA replication and repair, transcription, apoptosis and mitosis. We have previously shown that lamin B1 can contribute to the control of gene expression by tethering specific chromosomes [7] and the transcription factor, Oct-1 [5]. We found that in mouse embryonic fibroblasts (MEFs) lacking full length lamin B1, Oct-1 is no longer sequestered at the NE and is available to bind to its target sequences and regulate
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