KANK proteins

What are KANK proteins? KANKs are members of a protein family that is evolutionarily conserved from invertebrates to vertebrates. While Caenorhabditis elegans and Drosophila melanogaster express only one Kank gene, the vertebrate KANK family consists of four paralogs (KANK1–4) due to gene expansion and diversification. KANK1 is the largest protein, at ∼170 kDa. The other family members were identified via in silico analysis and are smaller, with KANK2 at ∼120 kDa, KANK3 ∼100 kDa and KANK4 ∼150 kDa. How did KANKs receive the name? The KANK gene was identified as a candidate tumor suppressor in a search for loss of heterozygosity in samples of human renal cell carcinoma. The search identified a novel gene, which is located on chromosome 9p, and displays high frequency of loss of heterozygosity. Based on the presence of ankyrin repeats at the carboxyl terminus and the identification of the gene in kidney tumor samples, Sarkar and colleagues termed the gene product KANK for kidney ankyrin repeat-containing protein. How are KANKs structured? The KANK proteins share similar protein structures, which include a unique KANK amino-terminal (KN) motif, variable numbers of central coiled-coil domains, and five carboxy-terminal ankyrin repeats (ANKs) (Figure 1A). Where are KANKs expressed? KANK family proteins have a cell-type-specific expression pattern in vivo. KANK1 is mainly found in epithelial cells, KANK2 primarily in mesenchymal cells such as fibroblasts, KANK3 exclusively in endothelial cells, and KANK4 in highly contractile cells such as pericytes, smooth muscle cells and mesangial cells. Staining of cells seeded on extracellular matrix revealed that KANKs localize to the rims of mature integrin-containing focal adhesions (FAs), termed the FA belt, and adjacent regions called cortical microtubule stabilizing complexes (CMSCs; Figure 1B). In contrast to the mammalian KANK3 expression pattern, studies regarding KANK3/NBP expression in zebrafish with a transgene encoding a GFP–NBP fusion protein revealed expression in the cytoplasm and cell–cell contacts from epiboly cells, on the basal side of neuronal cells, and in the notochord epithelium contacting the basement membrane. What are FAs and CMSCs? FAs are mature, integrin-based large macromolecular assemblies that link the extracellular matrix with the intracellular actomyosin cytoskeleton and transmit mechanical as well as biochemical information to and from the cell interior. Talin is an adaptor protein that binds integrin-β tails and actomyosin and plays a crucial role in activating integrins and mechanotransduction in FAs (Figure 1C). CMSC is a large proteinaceous machine that links microtubules via their plus-ends to the cell cortex at the leading edge of motile cells. The CMSC consists of several scaffolding proteins, including LL5β, ELKS, liprin α1, liprin β1 and KANK, and depletion of any CMSC component reduces the stability and density of cortical microtubules. KANKs link the CMSC to FAs via binding talin and liprin β1 (Figure 1C). What are the key functions of KANKs? KANKs play a key regulatory role for the crosstalk between FAs and CMSCs. KANKs, recruited to the FA belt via a direct association of the KANK KN motif with the talin rod R7 domain, curb the association of actomyosin, weaken integrin–ligand bonds, reduce cell migration speed and promote the translocation of integrins from the FA belt along actin filaments and to thin and elongated fibrillar adhesions in the cell center (Figure 1B,C). The KANK CC and ANK domains bind the CMSC component liprin β1 and the plus-end-directed motor protein KIF21A, respectively, and thereby couple microtubule dynamics at CMSCs with FA turnover by facilitating exocytosis of cargo, such as MT1-MMP, in the vicinity of FAs. The uncoupling of FAs and CMSCs — for example, by depleting KANKs or disrupting microtubule stability — leads to a dispersion of CMSCs at the cell cortex, loss of site-directed cargo secretion and the release of the RhoA GEF ARHGEF2/GEF-H1 from microtubules. This in turn results in increased RhoA-mediated actomyosin contractility, reinforcement of the stress fiber-associated FAs and decreased FA turnover. Thus, the dynamic assembly/disassembly of the talin–KANK–liprin β1 complex coordinates FA turnover and microtubule stability, which is key for cell movement (Figure 1C). Additional KANK functions identified include phosphorylation of KANK1 by growth factor signaling-induced Akt activation and its subsequent binding to 14-3-3 protein, which inhibits RhoA and stress fiber formation. KANK1 was also shown to compete with active Rac for IRSp53 binding and thereby block Rac-induced lamellipodia formation. The identification of nuclear localization as well as nuclear export signals in the KANK1 amino terminus and the association of KANK1 with β-catenin points to a role in Wnt signaling. KANK2 was shown to bind and sequester the members of the steroid receptor coactivator (SRC) family in the cytoplasm, which is why KANK2 is also called SRC-interacting protein (SIP). Estrogen-induced phosphorylation of KANK2 leads to their dissociation followed by SRC nuclear translocation, association with steroid receptors and gene transcription (Figure 1D). Are there any diseases associated with KANK proteins? Genetic studies by homozygosity mapping and whole-exome sequencing identified recessive mutations in KANK1, KANK2 and KANK4 in patients suffering from nephrotic syndrome. A family-based genetic linkage study in patients with cerebral palsy revealed a small genomic deletion spanning the KANK1 gene. Interestingly, studies of the mode of inheritance revealed that the disease is transmitted through the carrier fathers but not the carrier mothers, suggesting that the KANK1 gene is imprinted and expressed from the paternal allele. And a homozygous missense mutation in KANK2 disrupts its binding to SRC and is associated with keratoderma and woolly hair. Are KANKs essential for development? Genetic loss-of-function studies in mice have only been reported for the Kank4 gene so far, which revealed normal pre- and postnatal development and a function for artery growth upon ischemic stress. C. elegans and Drosophila harbor a single Kank ortholog. Whereas disruption of the dKank ortholog in flies is without phenotype, loss of the nematode ortholog, called VAB-19, leads to paralysis due to muscle detachment from the epidermis, axon protrusion defects and impaired basement membrane remodeling. In zebrafish, knockdown of Kank2 recapitulates a nephrotic syndrome-like phenotype and knockdown of Kank3 affects tissue morphogenesis and leads to embryonic lethality. What remains to be explored? Most of our knowledge originates from cell-based studies and human genetics. Loss-of-function studies in mice are restricted so far to KANK4. The extension to the other KANKs will provide important insights as to how individual KANKs orchestrate mammalian embryogenesis and whether they are redundant. So far, most reported KANK functions and binding partners are shared among mammalian KANK orthologs, although it is obvious that both KANK ortholog-specific binding partners and, hence, KANK-specific functions must exist. For example, SRC3/AIB1 seems to interact with KANK2 but not KANK1 (Guo et al.; unpublished data). Genetic studies in humans identified several disease entities associated with KANK family members. Besides these interesting genetic findings, whose molecular contexts are still largely elusive, KANKs also play a role in cancer biology. KANK1 for example, was shown to act as a tumor suppressor in renal cell carcinoma. This observation raises questions such as whether the tumor suppressor function is conserved in all epithelial tumors, how the tumor suppressive function is achieved at the molecular level and whether tumor development, growth as well as progression are affected by KANK1 and other KANK orthologs.