The entire ANKHD1 gene is about 147 Kb (start: 139781399 and end: 139929163 bp; orientation: plus strand). The ANKHD1 gene encodes for 4 transcript variants. The transcript variant 1 (start: 139781399 and end: 139919441 bp; orientation: plus strand; 34 exons; mRNA: 8233 bp) is the longest transcript and encodes the isoform 1 (2542 aa protein). The transcript variant 2 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 11 exons; mRNA: 2161 bp) uses an alternate in-frame splice site in the 5` coding region, lacks several exons, uses an alternate 3` terminal exon and encodes the isoform 2, which is shorter and presents a distinct C-terminus compared to isoform 1 (616 aa protein). Transcript variant 3 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 11 exons; mRNA: 2194 bp) lacks several exons, uses an alternate 3` terminal exon and encodes the isoform 3 that also has a distinct C-terminus compared to isoform 1 (627 aa protein). Transcrit variant 4 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 10 exons, mRNA: 2084 bp) also lacks several exons, uses an alternate 3` terminal exon, encodes the isoform 4 that has a distinct C-terminus compared to isoform 1 and is the shortest of all isoforms (581 aa protein). Additionally, ANKHD1-EIF4EBP3 is a readthrough transcript involving ANKHD1 and EIF4EBP3 genes, which encodes a protein that contains multiple ankyrin repeats, single KH-domain and a C-terminus with a portion from the EIF4EBP3 gene (start: 139781399 and end: 139929163 bp; orientation: plus strand; 36 exons, mRNA: 8349 bp; protein: 2617 aa). Data searched at GenBank NCBI (http://www.ncbi.nlm.nih.gov/genbank/) and UCSC Genome Browser (https://genome.ucsc.edu/).

The ANKHD1 protein has 4 isoforms. Isoforms 2, 3 and 4 are smaller and less characterized than isoform 1. Isoform 1 of ANKHD1 protein consists of 2542 amino acids with a molecular weight of 270 kDa, has 20 ankyrin repeats distributed in two blocks and a K homology (KH) domain in the C-terminal region. The schematic representation of ANKHD1 protein (isoform 1) is illustrated in Figure 1.

Ubiquitous.

ANKHD1 is predominantly found in the cytoplasm (Figure 2).

The ANKHD1 protein contains 20 ankyrin repeats, which are highly conserved structures. The presence of ankyrin repeats domains suggests that this protein integrates multiple signaling pathways and participates as scaffold for protein-protein interactions (Jernigan and Bordenstein, 2015). A large part of the knowledge regarding the functions of ANKHD1 was obtained by experiments using its orthologous protein Mask (multiple ankyrin repeats single KH domain). In Drosophila, Mask acts in the signal transduction pathway mediated by Cws (Corkscrew; orthologous of SHP2) (Smith, et al., 2002), participates in the Hippo signaling pathway activation through its association with Yki (Yorkie, orthologous of YAP1) (Sidor, et al., 2013, Sansores-Garcia, et al., 2013) and potentially regulates Hop signaling pathway (Hopscotch, orthologous of JAK) (Muller, et al., 2005). In humans, ANKHD1 associates with SHP2, however the biologic consequence of this protein interaction remains indeterminate (Traina, et al., 2006). ANKHD1 and YAP1 interaction has also been confirmed in human cells (HEK293 and DU145 cells) and modulates the Hippo signaling pathway activation, proliferation and cell cycle progression (Sidor, et al., 2013, Sansores-Garcia, et al., 2013, Machado-Neto, et al., 2014). ANKHD1 interacts with SIVA and modulates the microtubule dynamics, cell proliferation and migration of human leukemia cells (Machado-Neto, et al., 2015). In addition, ANKHD1 modulates p21 (CDKN1A) expression and cell cycle progression in multiple myeloma cells (Dhyani, et al., 2015, Dhyani, et al., 2012). In NT2 cells, inhibition of the small isoform of ANKHD1 that lacks the KH domain (also designed as VBARP; isoform 3) triggered caspase-3/7 activation (Miles, et al., 2005), however no similar effect on apoptosis was observed in the other cell lines inhibited for all other ANKHD1 isoforms (Machado-Neto, et al., 2014, Machado-Neto, et al., 2015, Dhyani, et al., 2012). A potential model for ANKHD1 cellular functions and protein interactions is summarized in Figure 3.

ANKHD1 shares high homology with ANKRD17 (ankyrin repeat domain 17), both orthologous proteins to Mask protein from Drosophila. ANKHD1 also shares high homology among different species (Table 1).

Recurrent mutations in the ANKHD1 gene are rare, 180 substitution missense, 13 substitution nonsense, 58 substitution synonymous, 1 insertion frameshift and 1 deletion frameshift mutations are reported in COSMIC (Catalogue of somatic mutations in cancer; http://cancer.sanger.ac.uk/cancergenome/projects/cosmic).