Stress-induced release of Oct-1 from the nuclear envelope is mediated by JNK phosphorylation of lamin B1.

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.

Nuclear envelope invaginations and cancer.

The nuclear envelope (NE) surrounds the nucleus and separates it from the cytoplasm. The NE is not a passive structural component, but rather contributes to various cellular processes such as genome organization, transcription, signaling, and stress responses. Although the NE is mostly a smooth surface, it also forms invaginations that can reach deep into the nucleoplasm and may even traverse the nucleus completely. Cancer cells are generally characterized by irregularities and invaginations of the NE that are of diagnostic and prognostic significance. In the current chapter, we describe the link between nuclear invaginations and irregularities with cancer and explore possible mechanistic roles they might have in tumorigenesis.

The nuclear envelope and its involvement in cellular stress responses.

The nuclear envelope is not only important for the structural integrity of the nucleus, but also involved in a number of cellular functions. It has been shown to be important for maintaining and controlling chromatin organization, sequestering transcription factors, replication, transcription and signalling. The nuclear envelope is thus important for development and differentiation, and some of its components are essential for cell viability. Among the many functions which are emerging for the nuclear envelope is its involvement in protecting the cell against different types of cellular stress. In the present paper, we review key findings which describe the roles of nuclear envelope components in responses to common types of stress conditions.

The induction of a nucleoplasmic reticulum by prelamin A accumulation requires CTP:phosphocholine cytidylyltransferase-α.

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.

Lamin B1 controls oxidative stress responses via Oct-1.

Interaction of lamins with chromatin and transcription factors regulate transcription. Oct-1 has previously been shown to colocalize partly with B-type lamins and is essential for transcriptional regulation of oxidative stress response genes. Using sequential extraction, co-immunoprecipitation (IP), fluorescence loss in photobleaching, and fluorescence resonance energy transfer, we confirm Oct-1-lamin B1 association at the nuclear periphery and show that this association is lost in Lmnb1(Delta/Delta) cells. We show that several Oct-1-dependent genes, including a subset involved in oxidative stress response, are dysregulated in Lmnb1(Delta/Delta) cells. Electrophoretic mobility shift assay and chromatin IP reveal that Oct-1 binds to the putative octamer-binding sequences of the dysregulated genes and that this activity is increased in cells lacking functional lamin B1. Like Oct1(-/-) cells, Lmnb1(Delta/Delta) cells have elevated levels of reactive oxygen species and are more susceptible to oxidative stress. Sequestration of Oct-1 at the nuclear periphery by lamin B1 may be a mechanism by which the nuclear envelope can regulate gene expression and contribute to the cellular response to stress, development, and aging.

Structure-function relationships of the extracellular domain of the autosomal dominant polycystic kidney disease-associated protein, polycystin-1.

Polycystin-1 (PC-1) is a member of a novel family of proteins that have a multidomain structure. Although the C-terminal intracellular segments have been extensively studied, mainly with respect to their putative involvement in cell signalling, the potential function of the extracellular domains has received less attention. Mutations in PC-1 result in autosomal dominant polycystic kidney disease (ADPKD) which is characterised by perturbation of transport resulting in fluid accumulation, cell proliferation and modification of the extracellular matrix. The possibility that the interaction of a component of the extracellular matrix or some external factor with PC-1 may be important in the initiation or progression of ADPKD cannot currently be ruled out. The purpose of this review is to assess current evidence for the function of the PC-1 extracellular domains, and their potential implications for ADPKD.

Removal of detergents from protein extracts using activated charcoal prior to immunological analysis.

The use of dextran-coated activated charcoal (DCC) powder to absorb solubilising detergents from cell lysates is described. Normal embryonic epithelial cells were lysed in the presence of sodium dodecyl sulphate (SDS). The detergent was then absorbed with DCC to facilitate analysis of polycystin-1 with antibody-based methods. Polycystin-1 is a membrane protein that is involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The adverse effect of SDS on antibody-polycystin-1 binding was studied and the improvement resulting from its removal demonstrated using enzyme-linked immunosorbent assays (ELISAs). The results indicate that DCC can be used in a simple manner to remove highly reactive membrane-solubilising reagents from protein mixtures prior to immunological analysis. This procedure may be relevant to a variety of other techniques that are normally affected by detergents.

Interaction of the leucine-rich repeats of polycystin-1 with extracellular matrix proteins: possible role in cell proliferation.

Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of approximately 460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid-derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependent reduction in the rate of proliferation. Cyst fluid-derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.