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The Ajuba protein has three highly correlated and tandem LIM domains at the C-terminus. It has not been found that Ajuba's LIM structure has DNA-binding activity and mainly mediates the interaction of various proteins. It has a preLIM domain rich in glycine and proline residues at the N-terminus. There is also a nuclear export domain (NES) in the preLIM domain that allows Ajuba proteins to shuttle back and forth between the nucleus and cytoplasm, while the LIM domain directs nuclear localization.
At present, research on the biological functions of Ajuba is mostly concentrated in mammals. Due to the presence of the NES functional structure, Ajuba can shuttle back and forth between the nucleus and the cytoplasm. In the nucleus, Ajuba protein mainly binds to various transcription factors and regulates the transcriptional activity of these transcription factors. In the cytoplasm, Ajuba generally binds to transmembrane proteins or signaling proteins to mediate a wide range of biological functions including regulation of cell cycle, cell adhesion and ligation, cell migration, cell proliferation and apoptosis, and cell differentiation.
Ajuba Protein Is Involved in Mediating Signaling Pathway Interactions
In P19 cells deficient in Ajuba, Ajuba acts as a nuclear receptor-specific co-repressor, negatively regulating retinoic acid signaling. During zebrafish heart development, Ajuba plays a central role in the regulation of SHF by linking the RA signal to Is1, a key transcriptional factor of cardiac precursor cells, and binding to sl1, which inhibits its transcriptional activity. In mammalian and Drosophila cells, Ajuba LIM/dJub interacts with LATS/Wts and WW45/Sav to inhibit signaling in the Hippo signaling pathway.
Rauskolb et al. found that Ajuba also binds to Warts to form a complex and inhibits the Hippo pathway by linking to cytoskeletal tension. Therefore, Ajuba acts as a negative regulator in the Hippo signaling pathway. The Sun GP and Irvine KD studies found that the Ajuba protein is a conserved link between JNK and Hippo signaling, and JNK increases Yki and YAP activity by promoting binding of the Ajuba family of proteins to Warts and LATS. Therefore, the Ajuba protein links the JNK signal transduction pathway to regulate Hippo signaling. Tanaka et al. found that Ajuba negatively regulates YAP activity through the LATS family in the NF2-Hippo pathway and inhibits proliferation of malignant mesothelioma cells (MM) via Hippo signaling.
Figure 1. Two mechanisms for Ajuba regulation of the Hippo pathway. (Gumbiner, et al. 2014)
Ajuba Is Involved in the Process of Cell Mitosis and Regulates Cell Cycle
Aurora-A kinase is a serine/threonine kinase that plays a key role in cell mitosis and meiotic cell division. Aurora-A kinase is overexpressed in a variety of cancers. Bai et al. found that the PreLIM domain of Ajuba binds to Aurora-A and leads to autophosphorylation of the Aurora-AC-terminal kinase domain. In addition, Ajuba's LIM domain competitively binds to the N-terminus of Aurora-A, thereby inhibiting the interaction between the N-terminus and C-terminus of Aurora-A.
Bai et al. also found that Aurora-A mutants (AurA-K250G and AurA-D294G/Y295G) could not bind to Ajuba. However, such mutants themselves lack the interaction between the N-terminus and the C-terminus, resulting in defects in the formation of the mitotic spindle, which arrests cell mitosis in the G2/M phase. This suggests that K250, D294/Y295 play a key role in the cell cycle progression of the direct interaction between Aurora-A and Ajuba and the Aurora-A complex.
Ajuba Is Involved in Cell Adhesion
Cell adhesion affects cell growth and fate and provides spatial clues to cell polarity within the tissue. The LIM domain protein Ajuba has been found to be present at the cell-cell contact site and can shuttle into the nucleus to participate in cell adhesion and thereby affect cell fate and growth. Studies have found that in primary keratinocytes, Ajuba recruits cadherin-dependent cell adhesion complexes by binding to α-catenin. Ajuba also interacts directly with F-actin. These data reveal that Ajuba acts as a new component in cadherin-mediated cell junctions. It also suggests that Ajuba may act as a bridge between the cadherin adhesion complex and the actin cytoskeleton, thus contributing to the formation or enhancement of cadherin-mediated cell adhesion.
According to the study of cancer cells, cancer cells have a lower adhesion between cell-cell and cell-matrix than normal cells, which is essential for the invasion and metastasis of cancer cells. Ajuba can enhance cell adhesion by combining with cadherin. Therefore, it can inhibit the invasion and metastasis of cancer cells.
Ajuba Is Involved in Cell Migration
Cell migration requires a sheet-like pseudopod that extends at the leading edge to form a binder complex. Both of these processes are regulated by signaling proteins recruited to the new adhesion site. Studies have found that cell migration in Ajuba-deficient mice is inhibited, with no abnormal attachment of extracellular matrix proteins, cell proliferation or integrin activation. Phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important regulator of the cytoskeletal organization during cell migration. A study found that the LIM protein Ajuba interacts with the active PI(4)P5 kinase (PIPKI) and the major enzymes in the synthetic PI(4,5)P2 to regulate the expression level of intracellular PI(4,5). In vitro, Ajuba significantly activates the enzymatic activity of PIPKI while inhibiting the activity of PIPKII. Therefore, in addition to its effect on Rac activity, Ajuba can also influence cell migration by modulating the synthesis of PI(4,5)P2 and directly activating PIPKI enzymatic activity.
Ajuba Is Involved in Transcriptional Regulation
Snail transcription factors are central to epithelial-mesenchymal transition (EMT) repressors and major regulators during normal embryonic development and tumor metastasis. The study found that the LIM protein Ajuba is a specific secondary repressor that binds to Snail's transcriptional repression and EMT induction. Using a Xenopus neural crest as a model of Snail or Slug-induced EMT in vivo, the researchers found that Ajuba is a specific co-repressor of Snail/Slug and also contributes to neural crest development. Through EMT, epithelial cells acquire higher interstitial phenotypes such as migration and invasion, anti-apoptosis, and ability to degrade extracellular matrices. Ajuba acts as a co-repressor of the transcription factor Snail in EMT transformation, inhibiting EMT transformation. Therefore, Ajuba can be used as a key molecule for EMT treatment in tumor metastasis.