KIBRA: In the brain and beyond

Abstract

In mammals, the KIBRA locus has been associated with memory performance and cognition by genome-wide single nucleotide polymorphism screening. Genetic studies in Drosophila and human cells have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in human tumorigenesis. Recent studies also indicated that KIBRA is involved in other physiological processes including cell polarity, membrane/vesicular trafficking, mitosis and cell migration. At the biochemical level, KIBRA protein is highly phosphorylated by various kinases in epithelial cells. Here, we discuss the updates concerning the function and regulation of KIBRA in the brain and beyond.

1. KIBRA gene and WWC family

The gene KIBRA was first described in 2003 and the name was given for its predominant mRNA expression in kidney and brain (1). The human KIBRA gene localizes on the positive strand of chromosome 5q34. The coding region encompasses about 180,000 base pairs and has 23 exons. The full-length transcripts express proteins with 1119 (isoform 1) or 1118 (isoform 2) or 1113 (isoform 3) amino acids due to alternative in-frame splice sites in the 3′ coding region. Human KIBRA contains two WW domains (a short protein module of approximately 40 amino acids that has two highly conserved tryptophans) at the N-terminus (amino acids 7-39 and 54-86). WW domains are responsible for recognizing proteins with proline rich motifs such as PPxY (x represents any amino acid). The C2 domain (amino acids 655-783) contains two four-stranded β-sheets that are responsible for a Ca²⁺-sensitive interaction with phospholipids (2). Additionally, KIBRA also contains several coiled-coil structures, a glutamic acid-rich domain, a class III PDZ (PSD95/Dlg/ZO-1) binding motif and an atypical protein kinase C (aPKC) binding region (Figures 1 and 2).

Fig. 1.

Sequence alignment (Clustal 2.1) and domain features of human WWC family proteins. The NCBI accession numbers for each protein are: NP_001155133 (KIBRA/WWC1), NP_079225 (WWC2) and AGV22437 (WWC3). Color legends: yellow for WW domains; green for potential coiled-coil domains; red for C2 domain; pink for glutamic-rich region; blue for PDZ-binding motif.

Fig. 2.

KIBRA/WWC1 orthologs and phosphorylation sites. Various domains are marked with different colors. The known phosphorylation sites and their corresponding kinases (with matched colors) are also indicated.

KIBRA (also known as WWC1) belongs to the WWC (WW and C2 domain containing) family, which comprises two additional highly similar paralogs, WWC2 and WWC3, in addition to KIBRA/WWC1 (Figure 1) (3). WWC2 and WWC3 share high structural similarity with KIBRA/WWC1 except that the glutamic acid-rich domain is specific for KIBRA/WWC1. Besides brain and kidney, WWC2 and WWC3 are preferentially expressed in thyroid, immune cells, reproductive tissues, liver and lung. The functions of WWC2 and WWC3 are not well studied yet.

The WWC family is evolutionarily conserved. KIBRA has been identified in many species ranging from insects to all vertebrates, but does not exist in yeast and worm (Figure 2). However, not all species express all three WWC family proteins. For example, lower organisms including Drosophila only have KIBRA. While fishes encode only two WWC genes, most other vertebrates including frog, rat and human have all three WWC members (3). Notably, due to a chromosomal translocation event in the evolution of the mouse lineage, Mus musculus expresses only KIBRA/WWC1 and WWC2 but lacks WWC3 (4). Whether there is functional interplay among the WWC proteins is almost completely unknown. However, a recent study showed that WWC2 expression is upregulated in the developing brain of the KIBRA knockout mice, indicating a possible compensatory function of these WWC family members (5).

So far, five transcription starting sites (TSS) have been identified in the region of the KIBRA gene (6). The TSS1b and TSS1c are located 153 and 415 bp upstream of the earlier annotated TSS1a, while the TSS2 and TSS3 are located in the first intron of KIBRA. The TSS1b and TSS1c are constitutively used in kidney and brain cells, resulting in transcripts for full-length KIBRA. The TSS2 and TSS3 are exclusively used in kidney cells, initiating transcripts for KIBRA isoforms without WW domains. Accordingly, their promoters, which are also located in the first intronic region, are specifically activated in kidney cells. The transcription factor 7-like 2 (TCF7L2) is believed to regulate KIBRA promoters, and binding sites for TCF7L2 have been identified near the promoters (6).

2. Expression patterns of KIBRA

KIBRA mRNA is highly enriched in human kidney, brain and testes (1). Gene expression studies and immunohistological staining have shown that KIBRA is expressed in memory-related regions of the brain, such as hippocampus and cortex, as well as in the cerebellum and the hypothalamus (7, 8). In the kidney, KIBRA is expressed in glomerular podocytes, tubules and the collecting ducts (9). In human normal breast tissue, KIBRA mRNA can be found at all stages of gland development and KIBRA protein has been detected in the luminal epithelium surrounding the ducts (10). In normal gastric tissue, KIBRA is expressed at the apical and cell-cell junction regions, but in gastric cancer tissue, increased expression of KIBRA can be detected not only in apical and junctional regions but also in the cytoplasm (11).

At the subcellular level, KIBRA was mainly cytoplasmic in green monkey kidney (CV1) cells (1). In hippocampal neurons, KIBRA shows a somatodendritic distribution with a perinuclear enrichment, and KIBRA is also a component of the postsynaptic density in rat brain (8). In breast cancer cells, although KIBRA is mainly present in the cytoplasmic fraction, it can also be detected in the nuclear fraction (12). KIBRA can also be detected in the heart and colon. In cultured cells, KIBRA protein is readily detected in epithelial cells of mammary, pancreatic and prostate origin (L. Z. and J.D., unpublished observations).

3. Binding partners of KIBRA

As described above, KIBRA protein has multiple binding motifs for interacting with other proteins to exert its functions. About twenty interacting partners of KIBRA have been identified in recent years. KIBRA was first identified by yeast two hybrid screens as a binding partner of human dendrin (1), which contains two PPxY motifs that bind to the WW domains of KIBRA. Another interacting partner, synaptopodin, also contains PPxY and sequence homology with dendrin in the PPxY surrounding area, thus also binding to the WW domains (9). Dendrin and synaptopodin are both actin-cytoskeleton proteins, so their binding to KIBRA is considered to be involved in cell polarity and motility, by KIBRA linking them to the polarity proteins (9). An example of the polarity proteins is the PALS1-associated tight junction protein (PATJ), which binds to the ADDV motif at the extreme C-terminus of KIBRA. Through its WW domains, KIBRA was also reported to bind to another PPXY motif-containing protein, discoidin domain receptor family member 1 (DDR1), an epithelial-specific collagen-activated receptor tyrosine kinase, in mammary epithelium (10, 13). The association between KIBRA and DDR1 was attenuated by collagen-induced DDR1 phosphorylation (10).

KIBRA specifically binds to the catalytic domain of PKC isoform zeta, but not to other PKC family members (15). The amino acids 953-996 of KIBRA are responsible for the PKCζ binding. PKMζ contains a catalytic domain similar to that of PKCζ but without the regulatory domain (16), so it is likely that it binds to KIBRA as well (9, 17). The constitutively active kinase PKMζ is exclusively expressed in the brain and is necessary for maintenance of long-term potentiating (18). Therefore, KIBRA may regulate memory through interacting with PKCζ/PKMζ. Additionally, the sequences between the WW domains and the C2 domain (amino acids 129-525) of KIBRA was shown to bind to Sec3, a component of Exocyst, thus mediating the aPKC-Exocyst interaction during migration of normal rat kidney (NRK) cells (19).

In a study with mass spectroscopic analysis of the dynein light chain 1 (DLC1)-associated proteins from breast cancer cells, KIBRA was found to interact with DLC1, a cytoskeleton signaling component, although the interacting site on KIBRA was not determined (12). In the same study, KIBRA was also demonstrated to associate with histone H3 via its glutamic acid-rich region, indicating a bridging function of KIBRA between DLC1 and chromatin. Since DLC1 is also known as estrogen receptor (ER)-binding protein and enhances ER transactivation, the DLC1-KIBRA-histone-3 binding presumably facilitates the ER-chromatin interaction. In another study, KIBRA was revealed to interact with sorting nexin-4 (SNX4), which regulates transportation from peripheral early endosomes to the juxtanuclear endocytic recycling compartment (20). SNX-4 coordinates endosome sorting through association with the minus end-directed microtubule motor dynein, which is mediated by KIBRA binding to both SNX4 and DLC1 (20).

Recent studies have identified KIBRA/kibra (KIBRA in human and mouse and kibra in Drosophila) as an upstream regulator of the Hippo signaling network which acts by binding to multiple signaling molecules in this pathway (21–24). In Drosophila, kibra complexes with tumor suppressors Merlin (Mer) and Expanded (Ex), two upstream regulators of the Hippo pathway (21–23). Ex contains three PPxY motifs and the RxPPxY motif which was shown to bind to the first WW domain of kibra (22). Mer also associates with kibra (21–23), and both N- and C-terminal part of kibra are sufficient to associate with Mer (22). Salvador (Sav) is one of the core components of the Hippo pathway functioning downstream of the kibra-Mer-Ex complex and contains WW domains for homodimerization (25). By yeast two hybrid assays and co-immunoprecipitation, it has been shown that the WW domains of kibra and Sav can interact with each other to form kibra/Sav heterodimer (23). In cultured Drosophila cells, kibra also co-immunoprecipitates with warts, another core member of the Hippo pathway (22) and the immunoglobulin domain-containing cell adhesion molecule Echinoid (26). The association between KIBRA and neurofibromatosis type 2 (NF2, ortholog of Drosophila Merlin) seems to be conserved in human cells (22,24,27). However, unlike the case with Drosophila, KIBRA does not associate with FERM domain containing 6 (FRMD6, ortholog of Expanded) in mammalian cells (22). Association between large tumor suppressor (Lats1/2, orthologs of Drosophila warts) kinases and KIBRA was also reported to be mediated through the WW domains of KIBRA and the PPxY motifs of Lats (24). Moreover, KIBRA was also shown to interact with protein tyrosine phosphatase, non-receptor type 14 (PTPN14) and angiomotin family proteins (AMOT) (28), all of which are known regulators of YAP phosphorylation and localization. Therefore, KIBRA associates with multiple upstream components of the Hippo pathway from Drosophila to human cells, suggesting that KIBRA functions as a signal integrator in the Hippo pathway and indicating crosstalk between KIBRA and other upstream complexes in the regulation of Hippo-YAP activity (28) (Figure 3).

Fig. 3.

KIBRA in Hippo pathway. (A) In Drosophila, kibra forms complexes with Mer and Ex and functions upstream of the Hpo-Sav complex; kibra also interacts with Wts and Pez (PTPN14). (B) In human cells, KIBRA associates with multiple proteins and regulates the Hippo-YAP signaling activity independent of Mst1/2. Arrows and blunted ends indicate positive (activation) and negative regulation (inhibition), respectively. Dashed arrow indicates unknown interaction.

Posttranslational modifications (phosphorylations), and thus regulation of KIBRA function, are mediated through an association with a group of kinases including aPKC (15) and its neuronal isoform PKMζ [13], citron kinase (CIT) (28), Aurora kinases (29), cyclin-dependent kinase 1 (CDK1) (30), extracellular signal-regulated kinases (ERK) and p90 ribosomal S6 kinase (RSK) (31) as well as their cellular counterplayers protein phosphatase 1 (PP1) (29), cell division cycle phosphatase 14 (CDC14) (30) and PTPN14 (28, 32) (see also section 7 below). In addition to these binding partners, several other molecules have also been reported to interact with KIBRA, such as the protein interacting with C-kinase 1 (PICK1), which regulates the major excitatory neurotransmitter receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in brains (5), KIAA0513, a marker in brain tissue for schizophrenia (33) and the axonal fasciculation regulator fasciculation and elongation protein zeta1 (FEZ1) (34) (Table 1).

Table 1.

Binding partners of KIBRA/kibra

Name	Binding domains (or other influences on binding)	In vitro/In vivo	Ref.
Dendrin	WW domains of KIBRA	In vitro (Y2H)	(1)
PKCζ	The catalytic domain of PKCζ; C-terminus of KIBRA	In vitro (Y2H)/In vivo (co-localization)	(15)
Histone H3; DLC1	Glutamic acid-rich region of KIBRA	In vitro (GST)/In vivo (IP)	(12)
SNX4	Not determined	In vitro (Y2H)/In vivo (co-localization)	(20)
DDR1	WW domains of KIBRA; the PPxY motif in DDR1	In vivo (IP)	(10)
PATJ Synaptopodin	The ADDV (PDZ) motif of KIBRA binds to the eighth PDZ domain of PATJ; WW domains of KIBRA bind to synaptopodin	In vitro (Y2H; GST)/In vivo (co-localization)	(9)
PKMζ	C-terminal region of KIBRA	In vivo (IP)	(9, 17)
Exocyst Sec 3	Amino acids 129-525 of KIBRA	In vitro (Y2H)/In vivo (IP)	(19)
Lats1/2	The N-terminal 86 amino acids of KIBRA and the PPxY motif in Lats2	In vivo (IP)	(24)
Aurora-A	Ser 539 phosphorylation of KIBRA and amino acids 354-403 of Aurora-A.	In vivo (IP)	(29)
PP1	The catalytic activity of PP1 is required for binding to KIBRA.	In vivo (IP)	(29)
CDC14A/B	CDK1 phosphorylation of KIBRAis involved	In vivo (IP)	(30)
RSK1/2	Phosphorylation of KIBRA is required for interacting with RSK1, but not RSK2	In vivo (IP)	(31)
FEZ1	Coiled-coil domain of KIBRA	In vitro (GST; Y2H)	(34)
PAR3/PAR6β	Not determined	In vivo (IP; co-localization)	(14)
Citron (CIT)	PPxY motif of C-terminus of CIT kinase with WW domains of KIBRA	In vivo (IP)	(28)
AMOT	Not determined	In vivo (IP)	(28)
PICK1	Not determined	In vitro (Y2H)/In vivo (IP; co-localization)	(5)
Merlin/(NF2); Expanded	The first WW domain of kibra and the ExRxPPxY motif	In vivo (IP)	(21–24, 27)
PTPN14 (Pez)	Not determined	In vitro (Y2H)/In vivo (IP)	(28,32)
Salvador (Sav)	WW domains of kibra and Sav	In vitro (Y2H)/In vivo (IP) (Drosophila)	(23)
Echinoid (Ed)	Intracellular domain of Ed	In vivo (IP) (Drosophila)	(26)

Abbreviations: Y2H: yeast two hybrid; IP: immunoprecipitation; GST: Glutathione-S-Transferase pulldown

4. KIBRA function in the brain and kidney

Since KIBRA’s discovery, most studies of KIBRA have been focused on its role in learning and memory and other neurological disorders (For a review see ref.(35)). KIBRA is mainly expressed in memory-related regions of the human and rat brains including cortex and hippocampus (7, 8). In the hippocampus, KIBRA is mainly distributed in the somatodendritic region and enriched at the postsynaptic density (8). A single-nucleotide polymorphism (SNP) rs17070145 within the ninth intron of KIBRA was implicated in human cognition from three independent, cognitively normal cohorts (7). The T-allele carriers (TT or CT) perform better than T-allele non-carriers (CC) in memory performance. Functional magnetic resonance imaging (fMRI) detected the allele-dependent differences in hippocampus activations during memory retrievals; the T-allele non-carriers needed more activation to reach the same level of epitope memory as T-allele carriers (7). Another study also replicated the KIBRA effect on episodic memory, but found the T allele carriers had increased hippocampal activation, suggesting that the enhanced memory performance of T allele carriers is due to hippocampal activity, but not the negative compensation (36). The role of the rs17070145 SNP on the cognitive performance of normal individuals has also been demonstrated by many other studies (37–42). However, non-replications have also been reported in some cohorts (43–45). These different conclusions may come from differences in genetic background (39), ages (46), genders and life style (44), disease status (38, 47) and tobacco uses (48). A recent meta-analysis including more than thirteen thousand subjects reported a significant association between rs17070145 and both episodic and working memory (49).

The molecular consequences of the identified intronic SNP on KIBRA expression and function are not clear. However, a recent report demonstrated an almost complete linkage disequilibrium between rs17070145 and two exonic SNPs (rs3822660G/T and rs3822659T/G) that lead to a replacement of two adjacent amino acids (M734I and S735A) within the KIBRA C2 domain (2). The encoded KIBRA variants display different binding preferences for phosphatidylinositols on endosomal vesicles, which may affect their specific physiological role in higher brain functions and in neurological diseases such as Alzheimer dementia or schizophrenia. Interestingly, a so far uncharacterized exonic SNP (rs139606423; R969W) within the KIBRA gene leads to a mutated binding region (and likely an altered binding affinity) for PKMζ, an important regulator of long term memory storage (50). Further studies will be necessary to analyze a putative linkage between rs139606423 and cognitive performance.

Evidence for association between KIBRA and memory performance also came from animal studies. The RhoA/ROCK/Rac pathway has been implicated in cognitive function (51, 52). This pathway is an upstream modulator of PKCζ and thus may alter the activity of KIBRA (53). Peripheral delivery of the ROCK inhibitor hydroxyfasudil was revealed to improve spatial learning and working memory in rats (54). More direct evidence for the involvement of KIBRA in episodic memory is that both hippocampal knock-down of KIBRA in rats and KIBRA knock-out in mice reduced learning and memory performance in spatial memory tasks, accompanied with decreased PKMζ level (17). PKMζ is a brain-specific protein kinase and is involved in memory maintenance (50). KIBRA stabilizes PKMζ and prevents it from proteasomal degradation by direct interaction between a short sequence motif near the C-terminus of KIBRA and the kinase, suggesting that KIBRA may affect memory maintenance by regulating synaptic PKMζ level (17). More recently, training rats in the T-maze task significantly increased the performance accuracy of rats and also increased the expression levels of KIBRA and PKMζ in the prefrontal cortex, supporting the notion that KIBRA and PKMζ is closely related to reference memory in rats (55). Additionally, KIBRA may regulate higher brain function by regulating AMPAR trafficking and synaptic plasticity (5). Adult KIBRA knockout mice showed reduced long-term potentiating and long-term depression as well as deficits in contextual fear learning and memory (5).

Associations between the KIBRA locus and several neuropsychiatric diseases were also evidenced. The first study on KIBRA and Alzheimer disease (AD) in 2009 revealed that the T allele of KIBRA rs17070145 was significantly associated with increased risk for very-late onset AD (OR = 2.89) (56). However, a later study showed the opposite conclusion; the T allele non-carriers showed increased risk of late onset AD in 2 cohorts (57). In the latter study, microarray assay on laser-captured microdissected neurons exhibited up-regulation of KIBRA in AD-affected brain regions, including the hippocampal, posterior cingulate and temporal cortex regions. The rs17070145 T allele non-carriers have reduced glucose metabolism in AD-affected regions in comparison to T allele carriers, as detected by positron emission tomography. In contrast, in an Asian cohort, the rs17070145 SNP was not associated with the late-onset AD but associated with the young AD patients (58).

In some patients, the KIBRA genotype affects memory differently compared to healthy subjects. For example, in patients with traumatic brain injury and subjective memory complaints, the rs17070145 T allele non-carriers perform better than T allele carriers (59, 60). This suggests that the C allele may have positive functions in pathological conditions. No association between KIBRA genetic polymorphism and mild cognitive impairment or recurrent depressive disorders was found (41, 61).

KIBRA is highly expressed in kidney, which points to a crucial role of this protein in renal functions (1). Indeed, a disturbed expression of KIBRA in kidney podocytes affects their migration activities and processes involved in cell polarity (9). Furthermore, patients suffering from focal segmental glomerulosclerosis (FSGS) display an upregulated KIBRA expression in glomeruli, indicating that KIBRA is crucial for normal kidney physiology (62).

5. KIBRA in cell polarity and trafficking

By linking the tight junction protein PATJ and cytoskeleton protein synaptopodin, KIBRA positively modulates the directional migration of podocytes (9). In migrating podocytes, KIBRA accumulates and co-localizes with PATJ and synaptopodin in the leading edge and modulates cell migration (9). This study also provided new information regarding the function of KIBRA: it serves as a linker molecule between polarity proteins (e.g. PATJ and aPKC) and components of the cytoskeleton (e.g. synaptopodin and dendrin) to regulate cell migration. Similarly, interaction between aPKC and Exocyst is required for proper cell migration in NRK cells and KIBRA mediates this interaction by linking aPKC and Sec3, a component of Exocyst (19). KIBRA knockdown in NRK cells inhibited aPKC localization at the leading edge and thus inhibited cell migration. In Drosophila, the apical Hippo pathway complex (including kibra) localizes to cell-cell-contacts and signals through hippo and warts to regulate the polarization of actin and promote migration (63). Kibra is also required for oocyte polarity in Drosophila (23).

KIBRA can negatively regulate cell polarity by suppressing apical exocytosis in epithelial cells. The partitioning defective 3 (PAR3)-aPKC-PAR6 complex plays fundamental roles in cell polarity (64). KIBRA is localized in the same position (apical domain and cell-cell junctions) with aPKC in polarized epithelial cells, and is directly associated with aPKC (64, 65). Knockdown of KIBRA expanded the apical domain of Madin-Darby canine kidney (MDCK) cysts, suggesting that KIBRA suppresses apical domain expansion during cyst formation (14). This process was elucidated to be independent of Hippo pathway (14). KIBRA also suppresses the formation of apical-containing vacuoles through enhanced de novo apical exocytosis (14). Interestingly, the abnormal phenotypes in KIBRA knockdown cells were rescued by aPKC inhibition, indicating that KIBRA regulates apical domain development by inhibiting the kinase activity of aPKC (14). The overexpression of KIBRA in epithelial cells failed, but the overexpression of the aPKC binding site of KIBRA delayed the re-establishment of cell-to-cell contacts in a calcium switch assay (14). Since KIBRA expression is highly enriched in the brain, it is surprising that the role of KIBRA in the polarity of neurons (which are highly polarized cells) has not been determined.

Recent studies also showed that KIBRA plays a role in vesicular trafficking. For example, KIBRA is involved in trafficking of AMPAR, which is the major excitatory neurotransmitter receptor in the brain, and which is assembled by four subunits (GluA 1-4) (66). KIBRA associates with AMPAR and its partner PICK1. KIBRA knockdown does not affect internalization of the AMPAR, but accelerates the rate of GluA recycling to the membrane, indicating that KIBRA helps retain AMPAR in cell plasma after internalization (5). Makuch et al. speculated that the loss of KIBRA can be compensated by the homologous protein WWC2, explaining the unaffected basal transmission and surface expression of AMPAR receptors in the KIBRA knockout mice (5). KIBRA is also necessary for transferrin receptor (TfnR) trafficking in HeLa cells (20). Although knockdown of KIBRA does not affect the internalization of TfnR, it retains TfnR in the endosomal sorting compartment and inhibits the trafficking of TfnR to the endosomal recycling compartment, thus increasing the lysosomal-mediated degradation of TfnR (20).

6. KIBRA in growth control and human cancer

Although numerous studies have defined the roles of KIBRA in the brain, its physiological function in non-neuronal cells is relatively less understood. In Drosophila, kibra was shown to function as a tumor suppressor that regulates the Hippo signaling pathway, which controls tissue growth and organ size (21–23). Kibra associates with Mer and Ex and directly binds to Hippo-Sav complex to regulate the Hippo signaling pathway. Loss of kibra results in imaginal disc overgrowth, oogenesis defects and increased target gene expression of Hippo signaling (23). Human KIBRA functions together with NF2 to stimulate Lats1/2 phosphorylation, thus inducing activation of the Hippo pathway to suppress the transcriptional activity of YAP (yes-associated protein), which is the downstream effector of Hippo signaling (23,24), indicating that the tumor suppressive function of KIBRA may be conserved in the mammalian system. Indeed, loss of KIBRA expression in immortalized breast epithelial cells results in epithelial-to-mesenchymal transition (EMT) features which are concomitant with decreased phosphorylation levels of Lats and YAP, but not mammalian sterile-20 like (Mst), and reduced expression of KIBRA in breast cancer specimens of Claudin-low subtypes correlates with poor prognosis (67).

The KIBRA promoter contains a well-defined CpG island (68). Hypermethylation in this region was detected in 70% of B-cell acute lymphocytic leukemias, but almost no methylation was found in common epithelial cancers including breast, colorectal, kidney, lung, and prostate (68). The reason for this highly cell type-specific inactivation of KIBRA is not known at this time. However, there is no obvious CpG island in the human WWC2 and WWC3 locus. Interestingly, epigenetic inactivation (downregulation) of KIBRA was shown to be correlated with malignant state of B-cell acute lymphocytic leukemia (68). Furthermore, KIBRA promoter methylation status was also revealed to be associated with poor prognosis of chronic lymphocytic leukemia patients, including high CD38 expression and immunoglobulin heavy chain variable genes (IGHV) unmutated status (69). However, a role of KIBRA in human cancer (including leukemia) development has not been firmly established. The KIBRA null-allele mice are available and these mice exhibit overall normal development and growth (5,17). It is very possible that WWC2 compensates for KIBRA function during development, thus the double knockouts of both KIBRA/WWC2 will be needed to elucidate the physiological function of KIBRA (and WWC2) during normal and potential cancer development.

Although the above studies implicate a tumor suppressive function of KIBRA, a very recent study reported that overexpression of KIBRA in low aPKC-expressing gastric cancer correlates with enhanced lymphatic invasion and poor prognosis (11), indicating the positive role of the KIBRA-aPKC axis in promoting the progression of gastric cancer. In addition, downregulation of KIBRA significantly reduced cell proliferation and motility in breast cancer cells (31). In line with these studies, several previous studies have also demonstrated the positive role of KIBRA in regulating cell migration and proliferation (9, 10, 12, 14, 19). Thus, the dual function (suppressive or promoting) of KIBRA in cell proliferation/migration may be cell or tissue-type specific and more systematic investigations are required for clarity.

7. KIBRA regulation/phosphorylation

KIBRA is a phosphoprotein and multiple kinases have been identified to phosphorylate KIBRA. aPKCζ interacts with and phosphorylates KIBRA at Ser 975 and Ser 978 in vitro. This phosphorylation does not influence the cellular localization of KIBRA, but it may regulate KIBRA dimerization (15). However, it is not known whether these phosphorylations occur in cells. Recent studies demonstrated that several members/regulators of the Hippo pathway were involved in mitotic-related processes (70–74) and regulated during mitosis (75–78). Interestingly, as an upstream regulator of Hippo signaling, KIBRA is also regulated during mitosis. KIBRA Ser 539 is the primary phosphorylation site for Aurora-A and Aurora-B kinases both in vitro and in vivo, and this phosphorylation plays a role in mitotic progression (29). Aurora phosphorylation of KIBRA is dephosphorylated by PP1 during mitotic exit. Since Aurora kinases and PP1 play important roles in mitotic-related events such as spindle assembly and centrosome formation (79–82), KIBRA may also be a component of the mitotic apparatus (29). Further studies showed that KIBRA is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of Lats2 on Ser 83 through activating Aurora-A. Knockdown of KIBRA causes mitotic abnormalities, including mitotic spindle defects and chromosome misalignment (83). It is possible that KIBRA-Aurora-Lats2 all regulate the activities of each other to control proper mitotic events during cell cycle progression (83). In addition to the Ser 539 site, KIBRA is also phosphorylated at two highly conserved serine residues (Ser 542 and Ser 931) by CDK1 during spindle-damaging agents-induced mitotic arrest (30). Elimination of CDK1-mediated phosphorylation of KIBRA promoted cell exit from Taxol-arrested G2/M phase, suggesting a role of KIBRA and its mitotic phosphorylation in spindle checkpoint activation (30). In yeast, the phosphatase Cdc14 triggers mitotic exit by antagonizing Cdk-mediated phosphorylation of their substrates (84). Interestingly, the human phosphatases CDC14A/B associate with and dephosphorylate the CDK1-mediated phosphorylation of KIBRA (30), however a role of KIBRA in mitotic exit has not been established. Mitotic phospho-regulation of KIBRA does not affect the Hippo-YAP signaling activity (29, 30) and is likely independent of the Hippo pathway.

Our very recent study showed that KIBRA is also phosphorylated by the ERK-RSK cascade. ERK1/2 phosphorylates KIBRA at Ser 548 both in vitro and in vivo, and this phosphorylation is required for KIBRA-mediated cell proliferation in breast cancer cells (31). Interestingly, previous reports indicated that KIBRA is required for collagen-induced ERK signaling activation (10) and KIBRA knockdown abolished the ERK activity in migrating NRK cells (19). It is not clear at this time (and will be interesting to explore) whether ERK phosphorylation is involved in regulating KIBRA-mediated ERK activation. RSK1/2 are critical downstream mediators of ERK1/2 kinases, and were shown to associate with and specifically phosphorylate KIBRA at Thr 929 and Ser 947 (31). RSK phosphorylation positively modulates KIBRA activity in both proliferation and migration in breast cancer cells (31). The mitotic phosphorylation sites (Ser 539 and Ser 931; except for Ser 542, which only exists in vertebrates) and ERK-RSK sites (Ser 548, Thr 929 and Ser 947) are evolutionarily conserved from Drosophila to human (29–31) (Figure 2) and all these sites also exist in both human WWC2 and WWC3 (Figure 4). The aPKC sites are less conserved (Figures 2 and 4). However, there is currently no published study concerning the phospho-regulation of WWC2 and WWC3.

Fig. 4.

Conservation of phosphorylation sites among human WWC family proteins. Sequences are downloaded from NCBI as in Figure 1. Blue marks the Aurora phosphorylation site; red marks the CDK1 sites; green highlights the ERK site; the RSK sites are in dark blue and the PKC sites are indicated with purple.

In addition to its regulation by phosphorylation, KIBRA expression is greatly induced in response to progestin in progesterone-responsive human breast cancer cell lines (10), suggesting that KIBRA is also a hormonal-related protein, although the biological significance of this induction/regulation is not known.

8. Conclusions and future directions

Many reports from both human and mouse genetics have firmly established KIBRA’s function in the brain. However, it remains elusive whether or how the other two paralogs WWC2 and WWC3 play a role in brain physiology. Furthermore, how KIBRA is regulated in the neurons is still unclear. For example, is there any KIBRA phosphorylation site playing a role in the neuron/brain? Moreover, more data are needed to confirm the association of KIBRA and AD or other brain-related disorders. Such studies may ultimately demonstrate KIBRA as a highly attractive target for the treatment of neurological diseases, including AD and dementia (35). Beyond the brain, KIBRA is also attractive for its function in cancer cell migration and proliferation. However, most conclusions are based on cell culture models and few studies involve animal and human patients.

Highlights.

1. KIBRA is a memory-associated protein

2. KIBRA also plays roles in migration, polarity and growth control in epithelial cells

3. KIBRA is phosphorylated by various kinases

4. KIBRA functions as an adaptor protein to exercise its functions by interacting with other proteins.

Abbreviations

AD

Alzheimer disease

AMPAR

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

CDC14A/B

cell division cycle 14A/B

CDK1

cyclin-dependent kinase 1

DDR1

discoidin domain receptor family 1

DLC1

dynein light chain 1

EMT

epithelial-to-mesenchymal transition

ERK

extracellular signal-regulated kinases

ER

estrogen receptor

FEZ1

fasciculation and elongation protein zeta1

FRMD6

FERM (Band 4.1, Ezrin, Radixin and Moesin) domain containing 6

MDCK

Madin-Darby canine kidney

Mst1/2

mammalian sterile-20 like 1/2

NF2

neurofibromatosis type 2

NRK

normal rat kidney

Lats1/2

large tumor suppressor kinase 1/2

PAR

partitioning defective

PATJ

PALS1(protein associated with lin-seven 1)-associated tight junction protein

PDZ

postsynaptic density 95 (PSD95)/disc large (Dlg)/zonula occludens-1 (ZO-1)

PICK1

protein interacting with C-kinase 1

aPKC

atypical protein kinase C

PKM

protein kinase M

PP1

protein phosphatase 1

PTPN14

protein tyrosine phosphatase, non-receptor type 14

RSK1/2

p90 ribosomal S6 kinase 1/2

SNP

single nucleotide polymorphism

SNX4

sorting nexin 4

TCF7L2

transcription factor 7-like 2

TfnR

transferrin receptor

WWC

WW and C2 domain containing

YAP

yes-associated protein