TLK2 gene consists of 31 exons and 30 introns. The gene maps to 17q23.2 and is 144,574 bp long (NCBI Reference Sequence : NC_000017.11 : 62470908-62615481).

TLK2 is 144,574 bp long and is on the plus strand. TLK2 gene has 31 exons (According to NCBI).

TLK2 produces 10 coding transcripts (3608 bp, 3512 bp, 3449 bp, 3227 bp, 1784 bp, 767 bp, 559 bp, 550 bp, 530 bp and 512 bp), 7 non-protein coding transcripts (578 bp, 513 bp, 3536 bp, 877 bp, 606 bp, 541 bp and 436 bp) and there is also an NMD (non-sense mediated decay) transcript (ENST00000583690.6; 496 bp) generated from the locus. (https://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000146872;r=17:62458658-62615481).

There are 2 reported pseudogenes of TLK2 which are: TLK2P1 (tousled like kinase 2 pseudogene 1) on chromosome 17 and TLK2P2 (tousled like kinase pseudogene 2) on chromosome 10 (https://www.ncbi.nlm.nih.gov/, 2020). TLK2P1 and TLK2P2 are ubiquitously expressed in whole blood, white blood cells, lymph node, brain, cortex, cerebellum, spinal cord, tibial nerve, heart, artery, skeletal muscle, small intestine, colon, adipocyte, kidney, liver, lung, spleen, stomach, esophagus, bladder, pancreas, thyroid, salivary gland, adrenal gland, pituitary, breast, skin, ovary, uterus, prostate and testis (https://www.genecards.org/cgi-bin/carddisp.pl?gene=TLK2P1&keywords=tlk2p1; https://www.genecards.org/cgi-bin/carddisp.pl?gene=TLK2P2&keywords=tlk2p2).

Tousled-like kinase 2 (TLK2), together with Tousled-like kinase 1 ( TLK1), belongs to Tousled-like kinases family of serine/threonine kinases (Silljé et al., 1999; Mortuza et al., 2018; Segura-Bayona and Stracker, 2019). As a cell cycle regulated enzyme, TLK2 exhibits its highest level of activity during S-phase (Silljé et al., 1999).
TLK2 is composed of an N-Terminal region harboring a nuclear localization signal (NLS), a middle region of helixes containing three putative coiled coil (CC) domains and a C-terminal protein kinase catalytic domain. The C-terminal region of the protein kinase domain is known to carry multiple potential phosphorylation sites in its carboxy-terminus (C-tail) (Groth et al., 2003; Krause et al., 2003; Klimovskaia et al., 2014; Mortuza et al., 2018).
TLK2 is activated through cis-autophosphorylation events in its kinase domain. These autophosphorylation events induce a conformational change which allows further phosphorylation in the C-tail of TLK2 (Mortuza et al., 2018). The monomeric TLK2 exhibits a negligible catalytic activity in comparison to its dimeric form (Mortuza et al., 2018; Segura-Bayona and Stracker, 2019).
Homodimerization of TLK2 or heterodimerization with TLK1 requires the presence of the first coiled coil (CC1) domain. Activated/phosphorylated dimers of TLK2 can display higher orders of oligomerization through further phosphorylation of the phosphor-sites in the loops joining the CC domains (Mortuza et al., 2018). This oligomerization can trigger activation and may also increase the enzymatic activity through recruiting additional TLK2 molecules. While the dimeric/oligomeric constructs of TLK2 are able to phosphorylate a specific substrate, ASF1A, the kinase domain alone do not display this property, suggesting that either the CC domains or their role in oligomerization is crucial for substrate recognition (Mortuza et al., 2018; Segura-Bayona and Stracker, 2019). Mechanism and role of other phosphorylations at the N terminus remain to be further investigated (Hornbeck et al., 2015; Segura-Bayona and Stracker, 2019).

TLK2 longest transcript encodes for 772 amino acids. Molecular weight is 87,661 Da.

TLK2 transcript is mostly expressed in testis but also in brain, eye, endocrine tissues, lung, proximal digestive tract, gastrointestinal tract, liver and gallbladder, pancreas, kidney, urinary bladder, female tissues, muscle tissues, adipose and soft tissue, skin, bone marrow, lymphoid tissues and blood. (https://www.proteinatlas.org/ENSG00000146872-TLK2/tissue).

TLK2 is a nuclear protein but its subcellular localization may change depending on the cell cycle. TLK2 colocalizes to cytoplasmic intermediate filaments during G1 phase and to the perinuclear region at S phase (Yamakawa et.al., 1997; Zhang et.al., 1999).

Cell Cycle and DNA Damage
Following its identification as a mammalian homologue of A. thaliana Tousled kinase, TLK2 has been linked to a wide-range of cellular functions (Sillje et al., 1999). TLK2 exhibits a constitutive expression pattern throughout the cell cycle whereas its kinase activity fluctuates during the cell cycle and peaking in the S-phase (Sillje et al., 1999). Inhibition of DNA-replication and induction of DNA-damage in mid-S phase cells with various agents (e.g. aphidicolin, hydroxyurea, cisplatin, bleomycin, topoisomerase inhibitors, ionizing radiation) substantially decrease TLK2 kinase activity (Sillje et al., 1999; Groth et al., 2003). These results suggest that there is a direct link between TLK2 activity and ongoing DNA-replication and this activity is inhibited as a result of stalled replication fork on damaged DNA. In support, TLK2 activity is associated with maintenance of replication fork integrity and chromatin assembly (Lee et al., 2018). siRNA-mediated knockdown of TLK2 reduces the rate of DNA replication fork progression, disrupt replication-coupled nucleosome assembly and result in accumulation of replication-dependent single-stranded DNA. Moreover, sustained depletion of TLK2 result in accumulation of replication-dependent DNA-damage which in turn cause a TP53 -mediated G1-S checkpoint arrest (Lee et al., 2018). However, how TLK2 promotes genome stability and its role in DNA damage response (DDR) remain to be fully elucidated. One possible mechanism to associate TLK2 activity with DDR is based on the fact that H3/H4 histone chaperone ASF1 is an interacting partner of TLKs (Sillje and Nigg, 2001). TLK-mediated phosphorylation of ASF1 positively regulates ASF1a protein levels, hinders its proteasomal degradation (Pilyugin et al., 2009) and stimulates its association with soluble histones and downstream chaperones, CAF-1 and HIRA (Klimovskaia et al., 2014). In yeast, ASF1 is implicated in maintenance of genome stability and checkpoint recovery through its direct interaction with CHEK2 (RAD53) (Emili et al., 2001; Hu et al., 2001), but in mammalian cells it is indirectly regulated by checkpoint kinases through activities of TLKs (Segura-Bayona and Stracker, 2019). Although a direct interaction of Checkpoint kinase 1 ( CHEK1) with TLK2 has not been demonstrated in proteomics studies (Segura-Bayona et al., 2017), its paralog TLK1 has been reported as a substrate of CHEK1. Phosphorylation of TLK1 at serine 695 (S695) in response to DNA-damage results in transient inhibition of TLK1 activity (Groth et al., 2003; Krause et al., 2003). This modulation of TLK1 activity by CHEK1 may be a key event for regulating histone binding capacity of ASF1 and to allow chromatin restoration during DNA-repair.
Interestingly, overexpression of TLK2 in T47D cells disrupt the CHEK1/2-mediated DDR signaling, resulting in a delayed DNA repair process, impaired G2/M checkpoint and an enhanced chromosome instability (Kim et al., 2016a). However, it has not yet been fully elucidated whether TLK2 is also directly regulated by CHEK1 or by heterodimerization with C-terminally phosphorylated TLK1 (Segura-Bayona and Stracker, 2019).
On the other hand, TLK2 controls chromatin restoration after DNA-damage during G2 arrest through histone chaperone ASF1A. In the absence of TLK2, improper chromatin restoration results in decreases expression of pro-mitotic genes such as; CCNB1 (Cyclin B1) and PLK1 which in turn compromised cellular competence to recover from cell cycle arrests induced by DNA-damage (Bruinsma et al., 2016).
The Drosophila genome includes a single Tousled-like kinase gene and the encoded Tlk plays dual function to prolong the G2-phase and to promote the G2 recovery via modulating the "p38a" (symbol provided by Flybase for Drosophila genes) activity. According to the proposed model, Tlk remains in an inactive form when its cellular level is low. Alternatively, Tlk kinase activity can be inactivated as a consequence of dATM/DCHEK1 activity triggered by a stress-induced DNA and/or histone damage. This inactive Tlk associates with "Tak1", "Hsc70-5" (Flybase) and EEF1A1. The resulting protein complex elevates p38a activity, which in turn causes a delay in G2 phase. On the other hand, under conditions where TLK activity is high, active Tlk promotes G2 recovery by decreasing p38a activity and increasing ASF1 activity (Liaw and Chian, 2019).
Cancer
TLK2 function has been linked to patient outcomes in a variety of human cancers including breast cancer, glioblastoma, uveal melanoma, cervical squamous cell carcinoma and endocervical adenocarcinoma (Kim et al., 2016b; Lin et al., 2018; Lee et al., 2018). Analysis of copy number, RNA-seq and survival data sets for breast tumors from The Cancer Genome Atlas Project (TCGA) showed that TLK2 is amplified in nearly 10% of ER-positive breast cancers and its resulting overexpression correlates with worse clinical outcome (Kim et al., 2016b). A phosphoproteomic analyses of TCGA breast cancer samples performed by The Clinical Proteomic Tumor Analysis Consortium (CPTAC) independently identified TLK2 as an amplicon-associated, highly phosphorylated kinase in luminal breast cancers (Mertins et al., 2016). Ectopic expression of TLK2 in T47D luminal breast cancer cell line, which normally possesses low TLK2 levels, results in increased invasion and cell migration and this aggressiveness reversed upon withdrawal of TLK2 expression (Kim et al., 2016b). The TLK2 overexpression-mediated invasiveness of breast cancer cells may be attributed to EGFR / SRC / PTK2 (FAK) signaling axis as TLK2 overexpression increases phosphorylation of EGFR, SRC and FAK. Conversely, siRNA-mediated knockdown of SRC, FAK or EGFR decreased the migration ability of TLK2-overexpressing T47D cells (Kim et al., 2016b).
shRNA targeting of TLK2 suppresses ESR1 (ERα), BCL2 and SKP2 protein levels, upregulates CDKN1B (p27), impairs cell cycle progression through G1/S border, decreases colony-forming ability, induces apoptosis and significantly improves progression-free survival in vivo (Kim et al., 2016b). Activation of SRC signaling axis by TLK2 and effects of this relation on cancer progression was also proved by studies performed with glioblastoma cells (Lin et al., 2018). TLK2 is upregulated in invasive glioblastoma samples in comparison to normal tissues and non-invasive glioblastoma samples, and this upregulation is associated with worse patient outcomes. Overexpression of TLK2 in glioblastoma cells increased cell growth, invasion and migration, while silencing TLK2 result in cell cycle arrest at G2/M checkpoint and ultimately induction of apoptosis. Expression analysis for markers of epithelial-to-mesenchymal transition indicated that TLK2 overexpression is associated with decreased levels of epithelial markers CDH1 (E-cadherin) and TJP1 (ZO-1), whereas mesenchymal marker vimentin is increased. On the other hand, TLK2 knockdown resulted in an opposite effect (Lin et al., 2018). In vivo analysis of TLK2 activity on cancer progression created a link between TLK2 knockdown and reduction in tumor growth and metastasis (Lin et al., 2018).
Development
In addition to its roles in replication, cell cycle regulation and maintenance of genome stability TLK2 function also implicated in mammalian development. The characterization studies performed with TLK2-defficient mice suggested that TLK2 is required for normal differentiation of trophoblast lineages. TLK2 deficiency in mice resulted in late embryonic lethality due to placental failure. Histological examination of the labyrinths of TLK2 deficient mice placenta indicate a disorganized structure composed of less mature and less differentiated trophoblasts. Taken together, these data suggest a requirement to TLK2 for proper placental development and function (Segura-Bayona et al., 2017).
This effect of TLK2 may be a consequence of its roles in transcriptional regulation (Bruinsma et al., 2016). TLK2 loss was also correlated with decreased histone chaperon ASF1 (Anti-Silencing Function 1) phosphorylation (Segura-Bayona et al., 2017).

TLK2 is conserved among species such as chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, fruit fly, C. elegans and frog (https://www.ncbi.nlm.nih.gov/).

Copy number variations and mutations are reported for TLK2.

De novo loss of function mutations are detected in patients with facial dysmorphisms and microcephaly (Lelieveld et al., 2016). De novo mutations (DNM) also detected in Autism spectrum disorders (ASD) (ORoak et al., 2011; Takata et al., 2018) and schizophrenia (Gulsuner et al., 2013). DNMs on the serine/threonin catalytic subunit of TLK2 are substitutions and listed as; p.(H493R), p.(H518R), p.(D629N), p.(R270) and c.(1786+1G>T) were detected in Intellectual Disability (ID) patients (Lelieveld et al., 2016).

Copy number increases (CNI) are seen in breast cancer patients (Kelemen et al., 2009; Kim et al., 2016b). Mutations of TLK2 are also reported for adenocortical carcinoma, bladder urothelial carcinoma, oligoastrocytoma, astrocytoma, breast invasive mixed mucinous carcinoma, breast invasive lobular carcinoma, breast invasive ductal carcinoma, cervical squamous cell carcinoma, colon adenocarcinoma, rectal adenocarcinoma, mucinous adenocarcinoma of the colon and rectum, glioblastoma mutiforme, head and neck squamous cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, serous ovarian cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, dedifferentiated liposarcoma, cutaneous melanoma, stomach adenocarcinoma, mucinous stomach adenocarcinoma, uterine carcinosarcoma / uterine malignant mixed mullerian tumor, uterine endometrioid carcinoma, uterine serous carcinoma/uterine papillary serous carcinoma, uterine mixed endometrial carcinoma (cBioPortal database) (Cerami et al., 2012; Gao et al., 2013).