CAMK2N1 Human

What is CAMK2N1 and what is its primary biological function?

CAMK2N1 (Calcium/calmodulin-dependent protein kinase II inhibitor 1), also known as CaMKII inhibitory protein alpha or CaMKIIN-alpha, is a potent and specific inhibitor of CaM-kinase II (CAMK2). This protein interacts with CAMK2B as part of the CAMK2N family and also interacts with CAMK2A in a calcium-dependent manner, requiring CAMK2A activation by Ca2+. Its primary function involves the regulation of calcium/calmodulin signaling pathways, which are critical for numerous cellular processes including cell differentiation, proliferation, and apoptosis . The human CAMK2N1 is a 78-amino acid protein with a molecular mass of approximately 10.9kDa when expressed recombinantly with an additional His-tag .

Where is CAMK2N1 located in the human genome and what is its structure?

CAMK2N1 is located on Chromosome 1 in the human genome. It can be found in the genomic sequence references NC_000001.11 (GRCh38.p14 Primary Assembly) and NC_000001.10 (GRCh37.p13 Primary Assembly) . The gene contains CpG islands in its promoter region and first exon, which makes it susceptible to epigenetic regulation through DNA methylation. The promoter region contains a large number of CG loci, with particularly significant methylation sites identified in the first amplicon of the gene . The recombinant human CAMK2N1 protein is typically produced as a single, non-glycosylated polypeptide chain containing 101 amino acids (including a 23 amino acid His-tag at the N-terminus) with the amino acid sequence: MGSSHHHHHH SSGLVPRGSH MGSMSEVLPY GDEKLSPYGD GGDVGQIFSC RLQDTNNFFG AGQNKRPPKL GQIGRSKRVV IEDDRIDDVL KNMTDKAPPG V .

How is CAMK2N1 expression regulated in normal and pathological conditions?

CAMK2N1 expression is tightly regulated through epigenetic mechanisms, particularly DNA methylation. In normal prostate epithelial cells (RWPE-1), CAMK2N1 shows low methylation levels (approximately 6.8%) and consequently higher expression. In contrast, prostate cancer cells demonstrate significantly higher methylation levels: 56.3% in LNCaP cells, 22.3% in DU145 cells, and 19.1% in PC-3 cells . This hypermethylation correlates with reduced expression of CAMK2N1, suggesting epigenetic silencing as a key regulatory mechanism. Interestingly, androgen receptor (AR) status appears to influence methylation patterns, with AR-positive LNCaP cells showing higher DNA methylation percentage than AR-negative DU145 and PC-3 cells . TCGA database analysis confirms that CAMK2N1 expression is reduced in prostate cancer tissues compared to normal prostate tissues, correlating with higher DNA methylation levels .

What methods are recommended for proper handling and storage of recombinant CAMK2N1 protein?

For optimal stability and functionality of recombinant CAMK2N1 protein in laboratory settings, researchers should store the protein at 4°C if the entire vial will be used within 2-4 weeks. For longer-term storage, the protein should be kept frozen at -20°C . To maximize stability during long-term storage, it is recommended to add a carrier protein such as 0.1% HSA (Human Serum Albumin) or BSA (Bovine Serum Albumin). Multiple freeze-thaw cycles should be strictly avoided as they can compromise protein integrity and activity . The recombinant protein is typically formulated as a sterile filtered colorless solution (0.25mg/ml) containing 20mM Tris-HCl buffer (pH 8.0), 0.15M NaCl, 20% glycerol, and 1mM DTT for optimal stability .

What methodologies are most effective for studying CAMK2N1 methylation patterns in cancer tissues?

For comprehensive analysis of CAMK2N1 methylation patterns in cancer tissues, researchers should employ multiple complementary approaches. Bisulfite sequencing (BS) provides detailed methylation analysis across all CpG sites in the promoter region, particularly focusing on the first amplicon which shows significant methylation differences between normal and cancer cells . This should be complemented with pyrosequencing for quantitative assessment of methylation at specific CG sites (particularly sites 4-8 in the first amplicon) which is especially suitable for FFPE tissue samples . Methylation-specific PCR following 5-Aza-CdR treatment allows researchers to establish causality between methylation and gene expression. Expression analysis through qRT-PCR and western blot should be performed in parallel to correlate methylation status with protein levels .

For clinical samples, researchers should analyze both the downstream and upstream regions of the transcription start site (TSS), as hypermethylation patterns differ significantly between these regions. In silico analysis using TCGA data can provide validation against larger patient cohorts and allow correlation with clinical parameters. When analyzing clinical samples, it is critical to include appropriate controls (such as benign prostatic hyperplasia for prostate cancer studies) and to stratify patients by clinical parameters including TNM stage, Gleason score, and PSA levels .

How does CAMK2N1 interact with DNMT1, and what experimental approaches best elucidate this relationship?

CAMK2N1 and DNMT1 (DNA methyltransferase 1) form a complex regulatory feedback loop in prostate cancer cells. DNMT1 mediates DNA hypermethylation of CAMK2N1, leading to its downregulation, while CAMK2N1 inhibits DNMT1 expression through suppression of the AKT or MEK/ERK signaling pathways . To investigate this relationship, researchers should employ a multi-faceted experimental approach.

First, genetic modification of DNMT1 through overexpression or knockdown should be performed to assess resulting changes in CAMK2N1 methylation and expression. Complementary to this, 5-Aza-CdR treatment can be used to inhibit DNA methylation, followed by assessment of both CAMK2N1 and DNMT1 expression . Researchers should also manipulate CAMK2N1 expression and analyze effects on DNMT1 levels. Signaling pathway analysis is crucial and should include examination of phosphorylated AKT and ERK following CAMK2N1 manipulation, potentially supplemented with pathway inhibitors to confirm mechanism specificity. Chromatin immunoprecipitation assays would provide direct evidence of DNMT1 binding to the CAMK2N1 promoter region.

What functional assays are recommended for investigating CAMK2N1's tumor suppressor role in cancer?

To thoroughly investigate CAMK2N1's tumor suppressor function in cancer, particularly prostate cancer, researchers should implement a comprehensive suite of functional assays. Wound healing, invasion, and migration assays provide critical insights into CAMK2N1's ability to inhibit cancer cell motility and invasiveness . These in vitro assays should be coupled with xenograft models in nude mice to validate tumor suppressive effects in vivo, measuring both tumor growth rates and metastatic potential .

For mechanistic studies, signaling pathway analysis through western blotting for phosphorylated AKT and ERK is essential to determine how CAMK2N1 exerts its tumor suppressive effects . Rescue experiments combining CAMK2N1 manipulation with DNMT1 overexpression can confirm their regulatory relationship. Cell cycle analysis and apoptosis assessment via flow cytometry should be performed to identify specific cellular processes affected by CAMK2N1. Experimental design should include multiple cell lines representing different AR status (LNCaP, DU145, PC-3) and comparison with normal prostate epithelial cells (RWPE-1) .

How does DNA hypermethylation of CAMK2N1 correlate with clinical parameters in cancer patients?

DNA hypermethylation of CAMK2N1 shows significant correlation with clinical parameters in prostate cancer patients. Analysis of FFPE tissue samples from prostate cancer patients reveals that specific CG sites, particularly site 4, demonstrate significantly higher methylation percentages in cancer tissues compared to benign prostatic hyperplasia (BPH) tissues . Patients with higher methylation percentages at site 4 exhibit higher TNM stages, higher Gleason scores, and elevated PSA levels, all indicators of more aggressive disease .

What signaling pathways mediate CAMK2N1's effects on cancer cell biology?

CAMK2N1 exerts its tumor suppressive effects through modulation of multiple signaling pathways, particularly the PI3K/AKT and MEK/ERK pathways . Experimental evidence indicates that CAMK2N1 inhibits these pathways, which in turn affects DNMT1 expression and activity. The ERK pathway specifically has been identified as an important signaling mechanism for DNA methylation regulation, with inhibition of MEK/ERK signaling reducing DNMT1 expression .

To investigate these pathways, researchers should employ western blotting for phosphorylated and total AKT and ERK following CAMK2N1 overexpression or knockdown. Pathway-specific inhibitors can help identify which signaling cascade predominates in different cellular contexts. The bidirectional relationship between CAMK2N1 and these pathways creates a regulatory feedback loop: CAMK2N1 suppresses AKT or ERK signaling, which reduces DNMT1 expression, consequently decreasing methylation of the CAMK2N1 promoter and increasing CAMK2N1 expression . This complex interplay necessitates careful experimental design with appropriate controls and time-course analyses to capture the dynamic nature of these relationships.

What are the key considerations when analyzing CpG island methylation in the CAMK2N1 promoter?

When analyzing CpG island methylation in the CAMK2N1 promoter, researchers must consider several critical factors. First, the promoter region contains two distinct CpG islands that are likely to be hypermethylated - one in the promoter region proper and another in the first exon . The first amplicon containing 22 CG sites shows significantly higher methylation percentages than other regions in both cancer and normal cells, indicating these CG sequences are key regulatory sites .

Methodologically, it's essential to analyze both the upstream and downstream regions relative to the transcription start site (TSS), as the search results indicate differential methylation patterns between these regions . When designing primers for bisulfite sequencing or pyrosequencing, researchers should target the region from CG14477205 to CG24294857 upstream of TSS, which shows the most significant hypermethylation in cancer samples . For clinical sample analysis, site 4 (CG22942704) appears particularly informative as it shows the strongest correlation with clinical parameters . Controls should include both technical controls for bisulfite conversion efficiency and biological controls (normal prostate epithelial cells or benign prostate tissues).

How should researchers design experiments to investigate the relationship between CAMK2N1 and androgen receptor signaling?

The search results indicate that AR-positive LNCaP cells demonstrate higher DNA methylation percentage of CAMK2N1 compared to AR-negative DU145 and PC-3 cells, suggesting a potential relationship between androgen receptor signaling and CAMK2N1 regulation . When designing experiments to investigate this relationship, researchers should include both AR-positive and AR-negative cell lines for comparative analyses.

A comprehensive experimental approach should include: (1) Treatment of AR-positive cells with androgens or anti-androgens followed by analysis of CAMK2N1 expression and methylation; (2) AR knockdown or overexpression studies to establish causality; (3) ChIP assays to investigate potential AR binding to CAMK2N1 regulatory regions; and (4) Analysis of patient samples stratified by AR status and correlation with response to androgen deprivation therapy. Time-course experiments are important to capture the dynamic nature of AR signaling. When analyzing clinical samples, patients should be stratified by AR status, and treatment history (particularly hormonal therapy) should be considered as a potentially confounding variable. This approach will elucidate whether AR signaling directly influences CAMK2N1 methylation and expression or if other mediators are involved.

What are the optimal conditions for using recombinant CAMK2N1 in in vitro enzymatic assays?

For optimal use of recombinant CAMK2N1 in in vitro enzymatic assays, researchers should consider several key parameters. The protein is typically available as a His-tagged recombinant product with greater than 90% purity as determined by SDS-PAGE . For enzymatic assays investigating CAMK2N1's inhibitory effect on CaM-kinase II, the reaction buffer should contain appropriate concentrations of calcium to facilitate the calcium-dependent interaction between CAMK2N1 and CAMK2A .

What approaches can be used to target CAMK2N1 methylation therapeutically?

More targeted approaches could include: (1) Development of CRISPR-based systems with targeted demethylation capabilities specific to the CAMK2N1 promoter; (2) Combination therapies of demethylating agents with pathway inhibitors affecting AKT or ERK signaling; (3) Targeting the CAMK2N1-DNMT1 feedback loop through specific modulators; and (4) Development of CAMK2N1 mimetic peptides based on functional domains that could bypass the need for gene reactivation.

Experimental validation of these approaches should include: in vitro assessment of CAMK2N1 re-expression and functional restoration; cell-based assays evaluating inhibition of invasion, migration, and proliferation; and in vivo testing using xenograft models. Therapeutic strategies should consider the complex regulatory relationships between CAMK2N1, DNMT1, and signaling pathways, as well as potential tissue-specific effects and the dynamic nature of epigenetic regulation .