Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein

Abstract

Cerebral cavernous malformations (CCMs) is a microvascular disorder in the central nervous system. Despite tremendous efforts, the causal genetic mutation in some CCM patients has not be identified, raising the possibility of an unknown CCM locus. The CCM2/MGC4607 gene has been identified as one of three known genes causing CCMs. In this report, we defined a total of 29 novel exons and 4 novel promoters in CCM2 genomic structure and subsequently identified a total of 50 new alternative spliced isoforms of CCM2 which eventually generated 22 novel protein isoforms. Genetic analysis of CCM2 isoforms revealed that the CCM2 isoforms can be classified into two groups based on their alternative promoters and alternative start codon exons. Our data demonstrated that CCM2 isoforms not only are specific in their subcellular compartmentation but also have distinct cellular expression patterns among various tissues and cells, indicating the pleiotropic cellular roles of CCM2 through their multiple isoforms. In fact, the complexity of the CCM2 genomic structure was reflected by the multiple layers of regulation of CCM2 expression patterns. At the transcriptional level, it is accomplished by alternative promoters, alternative splicing, and multiple transcriptional start sites and termination sites; while at the translational level, it is carried out with various cellular functions with a distinguishable CCM2 protein group pattern, specified abundance and composition of selective isoforms in a cell and tissue specific fashion. Through experimentation, we discovered a unique phosphotyrosine binding (PTB) domain, namely atypical phosphotyrosine binding (aPTB) domain. Some long CCM2 isoform proteins contain both classes of PTB domains, making them a dual PTB domain-containing protein. Both CCM1 and CCM3 can bind competitively to this aPTB domain, indicating CCM2 as the cornerstone for CCM signaling complex (CSC).

Emerging roles of CCM genes during tumorigenesis with potential application as novel biomarkers across major types of cancers

“…The relative expression of the CCM1 gene in homogenous cell populations from selected cell lines was also examined by qPCR (5). We found that compared to other cell lines, there was a much higher relative RNA expression level of the CCM1 gene in all cancer cell lines (5) and likewise, the same phenomena were also observed in the relative RNA expression levels of CCM2 isoforms (11). These data lead us to propose a potential role of the CSC in tumorigenesis in various tissues (5,11).…”

“…Expression of CCM genes at both the transcriptional and translational levels was confirmed through both qPCR and western blot analysis. Allele-specific real-time quantitative PCR (qPCR) assays were designed using boundary-spanning primer sets as previously described (11) and applied to quantify the RNA levels of the endogenously expressed CCM2 isoforms using Power SYBR Green Master Mix with ViiA 7 Real-Time PCR aystem (Applied Biosystems). TissueScan™ Real-Time PCR panels (HMRT100, 103, CSRT502) with Human β-actin control primer set (Origene) were used to determine the endogenous expression levels of CCM2 isoforms among the different tissues at the transcriptional level.…”

“…The relative expression level (2 -ΔCT ) was calculated from all samples and normalized to reference gene (β-actin); fold change (2 -ΔΔCT ) comparison was conducted by being further normalized to control groups (27). All experiments were performed with triplicates as described previously (11).…”

“…As a docking protein for both CCM1 and CCM3, CCM2 (isoform-100) has been reported to be upregulated in various cardiovascular conditions, indicating its role as a possible potent angiogenic factor (15). Furthermore, higher relative RNA expression levels of multiple CCM2 isoforms were also observed in all cancer cell lines, compared to normal primary cell lines (11), reinforcing the phenomena observed in CCM1 (5). CCM2 was found to be a key mediator of TrkA-dependent cell death in tumors, by coupling among TrkA signaling, caspase activation, and cell death.…”

“…As one of three key components of the CSC, CCM3 was initially identified as a tumor-associated apoptotic protein (7) which directly interacts with CCM2 within the CSC complex (8,9), suggesting that the CSC complex may be involved in tumorigenesis. CCM1 is ubiquitously expressed in various cells and tissues (5,10); likewise, almost all expressed isoforms of CCM2 are ubiquitously expressed in various cells and tissues (11), providing additional evidence for the potential involvement of the CSC in diverse cellular events in health and diseases, including cancer.…”