Phospho-LMNA (S22) Antibody
Description
Definition and Characteristics
Phospho-LMNA (S22) Antibody is a specialized immunological reagent that specifically recognizes and binds to the nuclear Lamin A/C protein only when it is phosphorylated at Serine 22 (S22). This antibody serves as a valuable tool for detecting and studying the phosphorylated form of nuclear lamins, which are intermediate filament proteins that form a meshwork underlying the inner nuclear membrane . The specificity of these antibodies is typically confirmed through various validation methods including ELISA using synthetic phosphorylated and non-phosphorylated peptides, phosphatase treatment of protein blots followed by western blotting, and comparative analyses in LMNA knockout cells .
Cellular Localization of Phosphorylated LMNA (S22)
Research has established that pS22-Lamin A/C exhibits a distinct localization pattern compared to non-phosphorylated Lamin A/C. Immunofluorescence microscopy studies in human fibroblasts have revealed that pS22-Lamin A/C signals primarily localize to the nuclear interior and are notably absent from the nuclear periphery . In contrast, non-phosphorylated Lamin A/C predominantly localizes to the nuclear periphery with only weak signals in the nuclear interior . This differential localization pattern persists throughout interphase, suggesting functional roles beyond mitotic nuclear envelope breakdown .
During mitosis, pS22-Lamin A/C signals increase dramatically, with strong signals observed throughout the cytoplasm when the nuclear envelope is absent during prophase and metaphase . This observation aligns with the established role of S22 phosphorylation in promoting Lamin A/C depolymerization in preparation for mitosis .
Role in Nuclear Lamin Dynamics
Phosphorylation of Lamin A/C at S22 plays a crucial role in regulating the polymerization and depolymerization dynamics of nuclear lamins. This post-translational modification affects the structural properties and localization of Lamin A/C in several ways:
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Increased nucleoplasmic localization: Phosphomimetic mutations of S22 lead to increased nucleoplasmic Lamin A .
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Enhanced mobility and dynamics: Phosphorylation at S22 facilitates faster movements of Lamin A/C both in the nucleoplasm and in the lamina .
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Increased solubility: Phosphomimetic S22 mutants exhibit higher solubility than wild-type Lamin A both in the lamina and in the nucleoplasm .
These effects are significant because they reveal how phosphorylation regulates Lamin A/C's structural and functional properties beyond simply facilitating nuclear envelope breakdown during mitosis .
Cell Cycle-Dependent Phosphorylation Patterns
Enhancer Binding and Chromatin Interactions
ChIP-seq experiments using Phospho-LMNA (S22) antibodies have revealed that pS22-Lamin A/C interacts with specific genomic sites that are characteristic of active enhancers . Importantly, these binding sites are located outside of lamina-associated domains (LADs), in sharp contrast to the binding pattern of non-phosphorylated Lamin A/C, which predominantly associates with LADs .
This differential genomic binding pattern suggests that pS22-Lamin A/C performs functions distinct from the traditional structural role of lamins at the nuclear periphery. The specific binding of pS22-Lamin A/C to active enhancers indicates a role in gene regulation and transcriptional activation .
Co-localization with Transcriptional Activators
Research has demonstrated that pS22-Lamin A/C binding sites frequently co-localize with binding sites for the transcriptional activator c-Jun . This co-binding pattern further supports the hypothesis that pS22-Lamin A/C is involved in transcriptional regulation, potentially facilitating the activity of enhancers and promoting gene expression .
| Feature | pS22-Lamin A/C Binding Sites | Non-phosphorylated Lamin A/C Binding Sites |
|---|---|---|
| Genomic Location | Outside LADs, at active enhancers | Predominantly at LADs |
| Co-binding Factors | Co-localization with c-Jun | Not specifically associated with transcriptional activators |
| Chromatin State | Associated with accessible chromatin | Associated with heterochromatin |
| Function | Potentially involved in gene activation | Involved in gene repression and nuclear organization |
Alterations in Disease States
In progeria patient fibroblasts, pS22-Lamin A/C binding patterns show significant alterations compared to normal fibroblasts. Some pS22-Lamin A/C binding sites are lost, while new binding sites emerge at abnormal locations . These changes in binding patterns are accompanied by increased histone acetylation, increased c-Jun binding, and upregulated expression of genes implicated in diseases associated with progeria, such as coronary artery diseases, hypertension, and cardiomegaly .
These findings suggest that alterations in pS22-Lamin A/C binding may contribute to the pathogenesis of progeria and potentially other laminopathies, providing new insights into the molecular mechanisms underlying these diseases .
Role in Cardiac Function
Research has established a link between Lamin A/C phosphorylation at S22 and cardiac function. Studies have shown that loss of phosphorylation at S22 significantly reduces the LMNA-mediated activation of peak sodium current (INa), suggesting a role in regulating cardiac conduction . This finding is particularly relevant because mutations in the LMNA gene are associated with cardiac conduction diseases (CCDs) and cardiomyopathy .
The observation that constitutive phosphorylation (using phosphomimetic mutations) partially restores reduced peak sodium current suggests that S22 could represent a potential therapeutic target in patients with LMNA-mediated cardiac conduction diseases . This functional link between phosphorylation of Lamin A/C at S22 and sodium channel function provides a molecular mechanism that may explain some aspects of laminopathy-associated cardiac pathologies .
Nuclear Morphology and Genome Integrity
Expression of non-phosphorylatable Lamin A/C mutants (T19A, S22A, and T19AS22A) has been shown to cause nuclear lamina defects, including invagination, micronucleus formation, and nuclear blebbing . These nuclear abnormalities highlight the importance of the phosphorylation/dephosphorylation-regulated polymerization/depolymerization of the nuclear lamina in maintaining genome integrity and optimal nuclear morphology .
Time-lapse imaging of cells expressing non-phosphorylatable Lamin A/C mutants has revealed abnormal reassembly patterns during mitotic exit, with detached filaments that fail to properly associate with chromosomes . These findings emphasize the critical role of properly regulated Lamin A/C phosphorylation in ensuring accurate nuclear envelope reassembly following mitosis .
Progeria and Aging-Related Diseases
The alterations in pS22-Lamin A/C binding observed in progeria patient fibroblasts provide insights into the molecular mechanisms underlying this premature aging disorder . The emergence of new pS22-Lamin A/C binding sites in normally quiescent loci, accompanied by increased histone acetylation and c-Jun binding, suggests dysregulated enhancer activity that may contribute to the disease phenotype .
These findings offer a novel perspective on how LMNA mutations cause progeria, shifting focus from the structural defects at the nuclear periphery to altered gene regulation mediated by phosphorylated Lamin A/C in the nuclear interior . This could potentially open new avenues for therapeutic interventions targeting the dysregulated enhancer activity in progeria and potentially other aging-related diseases .
Immunofluorescence Microscopy
Phospho-LMNA (S22) antibodies have been extensively used in immunofluorescence microscopy to visualize the localization of pS22-Lamin A/C within cells . These experiments have revealed the distinct localization pattern of pS22-Lamin A/C in the nuclear interior as opposed to the nuclear periphery, as well as its dynamic changes during the cell cycle .
Dual immunofluorescence with antibodies against pS22-Lamin A/C and pan-N-terminal Lamin A/C has provided comparative data on the differential localization of phosphorylated and non-phosphorylated forms of the protein . This approach has been instrumental in establishing the spatial segregation of different Lamin A/C populations within the nucleus .
Chromatin Immunoprecipitation and Genomic Studies
ChIP-seq experiments using Phospho-LMNA (S22) antibodies have mapped the genomic binding sites of pS22-Lamin A/C, revealing its association with active enhancers rather than LADs . These studies have transformed our understanding of Lamin A/C function, highlighting its role in gene regulation beyond its structural functions at the nuclear periphery .
The specificity of these antibodies has been crucial for distinguishing between the genomic interactions of phosphorylated and non-phosphorylated Lamin A/C, allowing researchers to delineate their distinct functions in the genome .
Western Blotting and Protein Analysis
Western blotting using Phospho-LMNA (S22) antibodies has been employed to quantify pS22-Lamin A/C levels during different phases of the cell cycle and in response to various experimental manipulations . These analyses have provided insights into the temporal dynamics of Lamin A/C phosphorylation and its regulation by kinases such as CDK1-Cyclin B1 .
The ability to specifically detect pS22-Lamin A/C in protein samples has facilitated studies on how this modification affects the biochemical properties of Lamin A/C, including its solubility and interaction with other proteins .
Product Specs
Target Background
- Lamin A-C interaction with Nestin and its role in tumor senescence. Nestin stabilizes lamin A-C to protect tumor cells from senescence. PMID: 30190500
- Among the 120 dilated cardiomyopathy patients, 13 (10.8%) harbored LMNA variants. A novel recurrent LMNA E115M variant was the most prevalent in familial DCM. PMID: 29386531
- Lamin A/C interacts with Notch signaling, thereby influencing cellular differentiation. Point mutations in LMNA could disrupt this interaction. PMID: 29040816
- Mutations in LMNA cause autosomal dominant severe heart disease, accounting for 10% of Dilated Cardiomyopathy. PMID: 29175975
- ZMPSTE24-dependent cleavage of prelamin A and the eight known disease-associated ZMPSTE24 missense mutations were examined. PMID: 29794150
- The LMNA-NTRK1 fusion was likely the molecular driver of tumorigenesis and metastasis in this patient. The observed effectiveness of crizotinib treatment provides clinical validation of this molecular target. PMID: 30134855
- Three heterozygous missense mutations were identified in unrelated patients - p. W520R (c.1558T > C), p.T528R (c.1583C > G), and p.R190P (c.569G > C). These variants are considered pathogenic, leading to isolated DCM with conduction defects or syndromic DCM forms with limb-girdle muscular dystrophy and Emery- Dreifuss muscular dystrophy. PMID: 29770364
- The functional integrity of lamin and nesprin-1 is thus required to modulate the FHOD1 activity and the inside-out mechanical coupling that tunes the cell internal stiffness to match that of its soft, physiological-like environment. PMID: 28455503
- The role of 1B and 2B domains in modulating elastic properties of lamin A. PMID: 27301336
- Progerin is upregulated in human dilated cardiomyopathy hearts and strongly correlates with left ventricular remodeling. PMID: 29702688
- Data indicate that patients with truncation mutations in LMNA (lamin A/C) had an earlier occurrence of cardiac conduction disturbance and low left ventricular ejection fraction than those with missense mutations. PMID: 29237675
- A novel truncating LMNA mutation associated with Cardiac conduction disorders and dilated cardiomyopathy was discovered in this family characterized by gender differences in clinical severity in LMNA carriers. PMID: 29628476
- We find no evidence for an elevated mutation rate in progerin-expressing cells. We conclude that the cellular defect in HGPS cells does not lie in the repair of DNA damage per se. PMID: 28477268
- Pathogenic gene mutations in LMNA and MYBPC3 alter RNA splicing and may have a role in heart disease. PMID: 28679633
- Patients with the heterozygous LMNA p.T10I mutation have distinct clinical features and significantly worse metabolic complications compared with other patients with atypical progeroid syndrome as well as patients with Hutchinson-Gilford progeria syndrome. PMID: 29267953
- Results suggest that lamin A/C might constitute a type of epithelial marker that better signifies EMT and MET in prostate cancer tissue. A decrease in lamin A/C expression in Gleason score (GS) 4 is likely associated with the EMT process, while the re-expression of lamin A/C in GS 5 is likely linked with MET. PMID: 29665450
- Using cardiomyocytes derived from human induced pluripotent stem cells carrying different LMNA mutations as a model for dilated cardiomyopathy, we demonstrate that PTC124 induces translational read-through over the premature stop codon and restores production of the full-length protein. PMID: 28754655
- This study represents a comprehensive report on the relative frequency of CMD in the UK population, indicating MDC1A as the most common CMD subtype (37.35%). PMID: 28688748
- In differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. PMID: 28854361
- A mutation in the gene encoding Lamin A/C (LMNAp.R331Q ) led to reduced maximal force development through secondary disease remodeling in patients suffering from dilated cardiomyopathy. PMID: 28436080
- In embryonic cells, upregulation of lamin A disrupts lamin C, which may influence gene expression. PMID: 27534416
- Our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in the regulation of NF-Y function in cell proliferation. PMID: 27793050
- Findings provide evidence that lamin A mutants (called progerin) activate the DNA damage response pathway and that dysregulation of this pathway may be responsible for the development of cardiovascular pathology in patients with Hutchinson-Gilford progeria syndrome. PMID: 28423660
- Type-2 familial partial lipodystrophy (FPLD2) is a rare autosomal dominant lipodystrophic disorder due to mutations in LMNA. PMID: 28408391
- The metabolic features of women with the Dunnigan variety of familial partial lipodystrophy, caused by several missense mutations of LMNA, are reported. PMID: 28443701
- UVA-induced progerin-lamin A complex formation was largely responsible for suppressing 53BP1-mediated NHEJ DSB repair activity. The present study is the first to demonstrate that UVA-induced progerin upregulation adversely affects 53BP1-mediated NHEJ DSB repair in human keratinocytes via progerin-lamin A complex formation. PMID: 28498430
- Suggest NF-YAs and lamin A expression levels as novel potential biomarkers useful to identify G1 endometrial carcinoma patients with a risk of recurrence. PMID: 27974701
- Finally, we demonstrate Lamins as the major factors in reliable miR-218 and miR-129 functions for breast cancer progression. Our findings uncover a new miRNA-mediated regulatory network for different Lamins and provide a potential therapeutic target for breast cancer. PMID: 29378184
- Data indicates that D243Gfs*4 LMNA as a mutation causing a severe form of cardiomyopathy with conduction defects, and suggest CX43 downregulation as a possible molecular mechanism leading to the conduction defects observed in mutation carriers. PMID: 29197877
- Two novel RNA isoforms of LMNA produced through alternative splicing. PMID: 28857661
- Lamin A/C is an autoantigen in Han Chinese patients with confirmed Sjogren's syndrome. Lamin A/C shares similar epitopes with U1RNP. PMID: 27835913
- It was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of the suspension state for tumor cells in tumor metastasis. PMID: 28919351
- In this report, we show that increased self-association propensity of mutant LA modulates the LA-LB1 interaction and precludes the formation of an otherwise uniform laminar network. Our results might highlight the role of homotypic and heterotypic interactions of LA in the pathogenesis of DCM and hence laminopathies in the broader sense. PMID: 28844980
- Familial partial lipodystrophy type 2 (FPLD2) is caused by an autosomal dominant mutation in the LMNA gene. FPLD2-adipocytes appear to accumulate markers of autophagy and catabolize triglycerides at higher levels than control adipocytes. PMID: 29108996
- We demonstrate that BAF is necessary to modulate prelamin A effects on chromatin structure. PMID: 26701887
- Dysmorphic nuclei in patients with an LMNA mutation correlate with the age of heart disease presentation. PMID: 29149195
- These results suggest that the nuclear lamins and progerin have marginal roles in the activation of the antioxidant Nrf2 response to arsenic and cadmium. PMID: 28229933
- We developed a proteomic analysis of plasma samples from a family showing a history of dilated cardiomyopathy caused by an LMNA mutation, which may lead to premature death or cardiac transplant. PMID: 27457270
- Exome sequencing of the proband revealed an extremely rare missense heterozygous variant c.1711_1712CG>TC; p.(Arg571Ser) in LMNA which was confirmed by Sanger sequencing in both the patients. Interestingly, the mutation had no effect on mRNA splicing or relative expression of lamin A or C mRNA and protein in the lymphoblasts. PMID: 28686329
- Case Report: pathogenic LMNA mutation gives a unifying diagnosis explaining arrhythmogenic right ventricular cardiomyopathy and Charcot-Marie-Tooth type 2B1 phenotypes. PMID: 27405450
- Standard Sanger sequencing of LMNA exon 11 DNA from blood-derived WBCs and cultured skin fibroblasts sequenced at passages 1, 3, and 8 detected differing progerin-producing mutations in the same nucleotide of the exon 11 intronic splice donor site (see online supplementary figure). PMID: 27920058
- The CNOT1-LMNA-Hedgehog signaling pathway axis exerts an oncogenic role in osteosarcoma progression, which could be a potential target for gene therapy. PMID: 28188704
- Pathogenic variants in the LMNA gene are responsible for nearly 10%-15% of Familial Dilated Cardiomyopathy cases. PMID: 27736720
- Low lamin A but not lamin C expression in pleural metastatic cells could represent a major actor in the development of metastasis, associated with epithelial to mesenchymal transition and could account for a pejorative factor correlated with a poor Performance status. PMID: 28806747
- These results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in Hutchinson-Gilford progeria syndrome. PMID: 28515154
- LmnA binds AIMP3 via its extreme C-terminus. Together, these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation. PMID: 28797100
- The R482W mutation results in a loss of function of differentiation-dependent lamin A binding to the MIR335 locus and epigenetic regulation of adipogenesis. PMID: 28751304
- Pathogenic variants of the LMNA gene were determined in nine families with familial partial lipodystrophy. PMID: 28641778
- The interaction of progerin with lamin A/C contributes to the development of the senescence phenotype of Hutchinson-Gilford progeria syndrome and aged cells. PMID: 27617860
- We expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that, contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages. PMID: 28056360
