Lamins are the major structural components of the nucleus and mutations in the human lamin A gene cause a number of genetic diseases collectively termed laminopathies. At the cellular level, lamin A mutations cause aberrant nuclear morphology and defects in nuclear functions such as the response to DNA damage. We have investigated the mechanism of depletion of a key damage sensor, ATR (Ataxia-telangiectasia-mutated-and-Rad3-related) kinase, in HeLa cells expressing lamin A mutants or lamin A shRNA. The degradation of ATR kinase in these cells was through the proteasomal pathway as it was reversed by the proteasomal inhibitor MG132. Expression of lamin A mutants or shRNA led to transcriptional activation of three ubiquitin ligase components, namely, RNF123 (ring finger protein 123), HECW2 (HECT domain ligase W2) and the F-box protein FBXW10. Ectopic expression of RNF123, HECW2 or FBXW10 directly resulted in proteasomal degradation of ATR kinase and the ring domain of RNF123 was required for this degradation. However, these ligases did not alter the stability of DNA-dependent protein kinase, which is not depleted upon lamin misexpression. Although degradation of ATR kinase was reversed by MG132, it was not affected by the nuclear export inhibitor, leptomycin B, suggesting that ATR kinase is degraded within the nucleus. Our findings indicate that lamin misexpression can lead to deleterious effects on the stability of the key DNA damage sensor, ATR kinase by upregulation of specific components of the ubiquitination pathway.

Lamins are components of the structural network in the nucleus and play a key role in nuclear organization and function. Mutations in the human lamin A gene cause a spectrum of genetic diseases that include muscular dystrophies, cardiomyopathy, lipodystrophy and premature ageing syndromes. Laminopathic cells display several abnormalities in nuclear morphology and function. We have carried out a comprehensive analysis of alterations in the protein profiles of HEK293T cells expressing a mutation in lamin A (Q294P) that causes Emery-Dreifuss muscular dystrophy, in comparison with cells expressing wild-type lamin A. Initially a total of 27 differentially expressed proteins were identified by quantitative two-dimensional gel electrophoresis followed by mass spectral analysis. Importantly, a 5-fold decrease in lamin B1 levels in mutant cells was observed by quantitative two-dimensional gel electrophoresis, which was supported by immunofluorescence analysis. Changes in levels of specific heat shock proteins, hnRNPs, DNA damage response proteins, metabolic enzymes and cytoskeletal proteins were also observed. Further detailed analysis of cell lysates by one-dimensional gel electrophoresis followed by high throughput mass spectrometry revealed 650 unique proteins from wild-type cells and 1494 unique proteins from mutant cells (with two or more than two peptide hits). Alterations in a number of proteins that are involved in chromatin structure, chromosome condensation, nucleosome assembly, epigenetic modifications, centromere organization, telomere length and DNA repair were observed. These data suggest that laminopathic cells exhibit characteristics of cellular stress and senescence, and provide new insights into the cellular basis of pathologies caused by laminopathic mutations.