Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest

Abstract

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16 ink4a , we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16 ink4a -mediated G 1 arrest. Reintroduction of lamin A, lamin C, or pRB restores p16 ink4a -responsiveness to Lmna ؊/؊ cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna ؊/؊ cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16 ink4a responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.A-type lamins are intermediate filament proteins that have been linked to the organization and maintenance of nuclear structure. In most differentiated tissues, A-type lamins (predominantly lamins A and C), along with lamin B, comprise the meshwork underlying the inner nuclear membrane known as the nuclear lamina (63). Unlike lamin C, lamin A contains a carboxy-terminal CaaX motif and must undergo a series of posttranslational modifications to form the mature lamin A (77). In particular, the cysteine of the CaaX motif in lamin A is isoprenylated, followed by cleavage of the aaX and carboxymethylation of the C-terminal cysteine (72). Lastly, a second cleavage event by Zmpste24 removes the last 15 amino acids to yield the mature lamin A (3, 13, 74).Mutations within the LMNA gene cause a variety of human disorders collectively known as laminopathies. These include progeria syndromes and dystrophies associated with skeletal muscle and adipose tissue (5,10,15,19,45,58). Mice lacking A-type lamins or deficient in processing lamin A develop skeletal and cardiac muscular dystrophies, thus confirming the importance of A-type lamins for muscle differentiation and/or maintenance (52, 65). The mechanism by which mutations in A-type lamins generate tissue-specific disorders is unknown. However, one model posits that lamin A/C regulates gene expression in differentiated tissue by coordinating the activity of key transcription factors.A-type lamins localize to p...

Suppression of Proliferative Defects Associated with Processing-defective Lamin A Mutants by hTERT or Inactivation of p53

“…Instead, expression of stably farnesylated lamin A protein was found to modestly enhance the proliferative life span of AG13353 fibroblasts. That this mutant differs from progerin in this assay is intriguing; although, we have previously observed distinct activities of progerin and L647R lamin A, most notably with respect to retinoblastoma (pRB) stabilization (Nitta et al, 2006). These differences in the regulation of pRB may partially underlie the enhanced proliferative life span associated with L647R-lamin A expression (see Discussion).…”

“…Given the ability of lamins to form stable polymers resulting from coiled-coil interactions, they have typically been ascribed a structural role in the nucleus (McKeon et al, 1986;Lammerding et al, 2004Lammerding et al, , 2006; however, a variety of regulatory roles have also been proposed (Spann et al, 1997;Spann et al, 2002;Frock et al, 2006;Ivorra et al, 2006;Nitta et al, 2006Nitta et al, , 2007Scaffidi and Misteli, 2008). The two classes of lamins, B-type and A-type (of which lamin A and lamin C are the most prominent), have different patterns of expression.…”

Constitutive Activity of JNK2α2 Is Dependent on a Unique Mechanism of MAPK Activation

“…Immunoprecipitations for 3ϫFLAG constructs were conducted in radioimmunoprecipitation assay buffer using 2 g of mouse anti-FLAG antibody (F3165; Sigma) bound to Gammabind G-Sepharose (GE Healthcare). The protein extracts were harvested and immunoblotted as described previously (16). The protein levels were quantitated using National Institutes of Health Image J.…”

“…Retroviral infections were carried out as previously described except that the Phoenix-Ampho cell lines were used instead of HEK293 cells (16). 36 h after infection of the U87-MG cells, the infected cells were selected by culturing in selective medium containing 500 g/ml of hygromycin-B for 2 days.…”

“…Protein Analysis and Immunoprecipitation-Protein extracts from total cells were harvested as previously described (16). 50 g of total protein was separated by denaturing electrophoresis.…”