TBC1 domain containing kinase (TBCK), also known as hematopoietic stem and progenitor cells 302 (HSPC302) or Mammalian Gene Collection 16169 (MGC16169), was initially discovered in CD34⁺ hematopoietic stem cells. It was then reported in the NEDO human cDNA sequencing project by researchers at the University of Tokyo (1,2). At that time, TBCK was considered a hypothetic protein with unknown functions. Since then, several groups have provided mRNA and peptide data of TBCK in different cell types. Resing et al (3) in 2004 identified 5130 proteins in the K562 cell line with high throughput shotgun proteomics, including two peptides of TBCK; Tanner et al (4) in 2007 also identified the expression of TBCK in HEK293 cell line with peptide mass spectrometry.

It has been shown that TBCK comprised three putative structural domains: S_TKc, TBC (Tre-2, Bub2 and Cdc16) and RHOD_Kc (5,6). TBCK included two types of alternatively spliced isoforms (long TBCK and short TBCK). Long TBCK comprised all the three conserved motifs, while short TBCK only comprised TBC and RHOD_Kc domains (Fig. 1A and B). At present, most of the TBCK-related research focuses on the relationship between TBCK gene mutations and neuronal development disorders. Our previous results have demonstrated TBCK's expression in a cell type-specific manner. The proteins produced by the two alternative isoforms contribute to differential functions of TBCK. In the following section, we will summarize the detailed gene structure, protein expression of TBCK and the important roles of TBCK in human diseases including cancers.

Although there is a long way to go to fully understand the function of TBCK, recent research indicates that TBCK plays an important role in brain development. Mutations to the TBCK gene could cause neurological developmental disorders. Until now, a total of 17 mutations were reported to be associated with neurodevelopmental diseases (Fig. 1A and Table II).

Most of the mutations were nonsense mutations, generating premature stop codons. After categorization, it can be found that 73.3% (11/15) of mutations were located in the region containing the first two domains, affecting the translation of full-length TBCK. It is worth noting that the two missense mutations happened in the RabGap domain, indicating that the RabGap domain might be the most important functional unit for proper brain development. However, the underlying molecular mechanisms still remain unknown.

Alazami et al (27) identified 69 genes related to neurogenic diseases through whole exome sequencing of 143 multiplex consanguineous families, of which an insertion mutation at 1709ntin the TBCK coding region was verified to cause a frameshift and further influence disease progression. This insertion was also detected in 13 individuals from nine unrelated families, likely being pathogenic variants of TBCK. Eight other mutations of the TBCK gene were reported to be the main cause of mental retardation and hypotonic syndrome, and L-type leucine-mediated activation of the mTOR signaling pathway helped alleviate related symptoms (28). In the meantime, another group verified two novel mutations of TBCK genes (c.1363A>T [p.Lys455*] and c.1532G>A [p.Arg511His) via whole-exome sequencing of infants with encephalopathy in 4 unrelated families, of which the former mutation would induce a stop codon and lead to the deletion of long TBCK, while the latter mutation was located in the TBC conserved domain and might affect the RabGap activity of TBCK (15). Unlike the overgrowth of the brain caused by mTOR pathway disorders, a gradually decrease of the brain volume of infants with encephalopathy would be caused by TBCK deficiency (29). Furthermore, six more mutations (either resulting in nonsense or frameshift) affecting the TBCK expression were reported by six different groups (Table II) (30–35).

It should be noted that four common mutations have been reported in different patients from at least two different groups (Table II): c.1897+1G>A (27,28); c.1652T>C (28,36); c.803_806delTGAA (28,29); and c.376C>T (15,28,37). All of the mutations would ablate the expression of full-length TBCK and cause TBCK-related developmental and neurological diseases. However, TBCK function has been poorly explored. Previous research shows that TBCK played a role in cell growth and actin organization by enhancing the signaling pathways of mammalian target of rapamycin (mTOR), presumably at a transcriptional or post-transcriptional level (5). Besides, TBCK deficiency would disturb activation of the mTOR complex 1 (mTORC1), thus, affecting the autophagy process and further leading to autophagosomal-lysosomal dysfunction (37). Nevertheless, does TBCK directly or indirectly affect the mTOR signaling pathway? Which domain contributes the most? What are the binding partners of TBCK? These open questions await further studies.

TBCK was expressed universally in almost all human tissues, except a relatively low expression in heart, brain, skeletal muscle, and peripheral blood leukocytes (data not shown). Besides, TBCK was proven to be down-regulated in 55.6% of paired gastric carcinoma and 75.0% pair-matched esophageal carcinomas. Overexpression of TBCK in HeLa cells could remarkably inhibit cell growth and arrest cells at S phase, which was indicative of tumor suppressive function (6). After analyzing the clinical information collected from TCGA, the five-year survival rates for patients with high-level TBCK was significantly higher than that of patients with low-level TBCK in renal cancer (P=3.20E-4) and pancreatic cancer (P=3.67E-2). A similar phenomenon could be found in breast cancer (P=2.50E-3) and lung cancer (P=4.70E-13) (Fig. 1D) using Kaplan-Meier Plotter database [https://kmplot.com/analysis/] (38). This implied that TBCK might also possess the potential to be a viable prognosis marker for treatment of some cancer types.

However, TBCK might also exhibit tumor-promoting functions in certain cancer types. Based on the Oncomine database (Fig. 1E), it has been shown that TBCK exhibit the tumor-promoting functions in leukemia, lymphoma, liver cancer and sarcoma, in addition, individual experiments also validated that exhibit the functions in squamous cell carcinoma and renal cancers (11,39). In a human kinase mapping study using the entire kinome siRNA library targeting over 600 related genes, TBCK-specific RNAi decreased the phosphorylation of ERK1/2 and increased the phosphorylation of STAT3. TBCK was further proven to selectively support coupling of active EGFR to ERK1/2 regulation (11). A very recent study on TBCK showed that TBCK was a direct target of miR-1208, and that the miR-1208/TBCK interaction had an important role in the regulation of apoptosis, as well as in the enhancement of cisplatin or TRAIL sensitivities in renal cancer cells (39). However, how TBCK involved in both tumor promotion and inhibition in different cancer types is unknown, and requires further investigation.

Previous results indicated that the eukaryotic protein kinase comprised of 12 essential conserved subdomains to maintain its kinase activity (40). Due to its lack of two important motifs (GXGXXG motif and VAIK motif) responsible for ATP binding, and the replacement of those motifs with mutated HRD motif that was essential for catalytic activity, TBCK was considered a pseudokinase (5,41,42). Itis implied that TBCK might phosphorylate the ERK1/2 protein (11). However, further direct kinase assays should be performed to clarify whether TBCK has kinase activity or not. The positive answer also provides evidence for differential functions between long and short isoforms of TBCK (6).

TBC domain-containing proteins usually function as a RabGap (Rab GTPase-activating protein) to negatively regulate Rab functions through accelerating GTP hydrolysis via a ‘dual-finger’ mechanism (13,43–45). Although the crystallographic structure of TBCK has not been reported, Chong et al (15) generated a homology model of the TBC1 domain of TBCK using DeepView and the SwissModel server (46) and uncovered a structural impact of the disease-causing amino acid substitution (p.Arg511His). Other reports also demonstrated that the TBC domain in TBCK included the key conserved amino acid residues required for RabGAP activity in functional RabGAPs (5,13). However, the direct substrate of TBCK was failed to be identified in a systematical screening for target Rabs (60 Rab proteins) of TBC domain-containing proteins (40 proteins including TBCK) based on their Rab-binding activity (14). Thus, it is necessary to carry out critical experiments to figure out the physiological target of TBCK, which shall provide direct evidence for the RabGap activity of TBCK.

In addition, previous studies have shown that TBCK mutations would cause neurogenetic disorders. The mTOR pathway and mTOR-mediated autophagy might play important roles in such processes (28,37). However, it is still unclear how TBCK affects the mTOR signaling pathway, what the interacting proteins of TBCK are and whether there are other pathways involved remains unsolved. Our current research has uncovered 17 candidate proteins of TBCK using RNAi-mediated TBCK silencing in combination with 2-DE-DIGE assays. These candidates played important roles in multiple activities, such as protein folding, post-translational modification, and the cytoskeleton etc. (data not shown). More work on the mechanism of action needs to be completed in order to clearly clarify the roles of TBCK in neurogenetic disorders and tumor development.

An important finding for TBCK function in recent years was that TBCK functions as a candidate RabGAP. Deleterious mutations of TBCK would ablate the function of TBCK and cause severe infantile syndromic encephalopathy or other neurogenetic disorders. These mutation sites were found in the whole exons covering three conserved domains. Abnormal function of TBCK would destroy the mTOR signaling pathway and its mTOR-mediated autophagy process, which was considered the major cause of TBCK-related neurogenetic disorders. In addition, two types of TBCK isoforms were verified, and the kinase domain might account for the functional differences among TBCK isoforms. Limited research also suggested that the distribution of TBCK was cell cycle-dependent, and the role of TBCK in tumors was cell line-dependent. Overall, the function of TBCK is poorly explored and awaits further investigation.