Alternative splicing of the Ctk transcript gives rise to two Ctk isoforms, isoform 1 and isoform 2 of 56 kDa and 52 kDa, respectively. The 56 kDa isoform 1 (p56Ctk) contains an additional 41-residue segment at the N-terminus. Northern blot analyses demonstrated that the (p56Ctk) isoform 1 is expressed in normal colon cells and hematopoietic cells of megakaryocyte lineage while the 52 kDa Ctk isoform (p52Ctk) is expressed in brain cells [(Avraham et al. 1995; Bennett et al. 1994; Kuo et al. 1994) and reviewed in (Cheng et al. 2006; Advani et al. 2015)]. Whether and how the two isoforms differ in their regulatory properties and functions remain unknown.
Gene Structure and Isoforms of Ctk
Sequence features of the humanCtkgene. Refer to Fig. 3 for further information of the Ctk genes, the transcripts generated from the gene, and the different isoforms of Ctk protein encoded by the transcripts
Promoter or exon
Contains a CpG island of 1142 base pairs with 74.5% GC content and an observed-to-expected CpG ratio of 92%
Generation of Transcripts 1 and 2, which govern the expression of the 56 kDa and 52 kDa isoforms (Isoforms 1 and 2) of Ctk
Lacks TATA box
Contains a GC-rich box of 270 bp upstream of the putative promoter region
The putative transcriptional regulatory sequences, such as GATA-1, Sp1, APRE, and APRE1 motifs are upstream of the TSS and the GC-rich box (Avraham et al. 1995)
Upstream non-canonical promoter
Contains CpG islands of 427 base pairs with 66.3% GC content and an observed-to-expected CpG ratio of 82%
Generation of Transcript 3, which can potentially direct the expression of Isoform 3 of Ctk. There is no documented evidence supporting the expression of Isoform 3
Contains (i) the 5′-untranslated sequence and (ii) the transcription start site (TSS) located 360 bp upstream of the canonical translation initiation site (in exon 2)
Transcript 1 containing exon 1 and the other 12 exons can undergo alternative splicing to generate Transcript 2
Transcript 2 lacks parts of exon 1 and exon 2 and contains a different translational initiation site. The resultant isoform 2 lacks the N-terminal 41 residue-segment of isoform 1
Contains translation initiation sites to direct expression of Isoforms 1 and 2 of Ctk
Encode the SH3 domain
Encode the SH2 domain
Encode the kinase domain
UCSC Genome Browser and Ensembl databases reported the existence of three mRNA transcripts of Ctk genes (Fig. 3b). Both transcripts 1 and 2 are generated by RNA polymerase II-mediated transcription using the canonical promoter (Fig. 3). Transcript 1 encodes the 56 kDa isoform 1, p56Ctk of 507 amino acid residues, which is expressed in multiple nonneuronal cells (Chow et al. 1994). Transcript 2, encoding the isoform 2, p52Ctk, which lacks the N-terminal 41 amino acid residue segment of isoform 1, is generated by alternative splicing. p52Ctk is expressed exclusively in neurons (Kuo et al. 1994; Kuo et al. 1997). Transcript 3 encoding isoform 3 has been documented in the UCSC Genome Browser. It contains a putative noncanonical promoter. There is no experimental evidence confirming the existence of isoform 3 of Ctk protein encoded by transcript 3 (Fig. 3b). Previous studies by Nakayama et al. demonstrated that the N-terminal region of p56Ctk contains structural features targeting it to the nucleus to phosphorylate nuclear protein substrates and inhibit cell proliferation in HeLa and Cos-1 cells (Nakayama et al. 2006). Additionally, two groups of researchers independently reported the presence of p56Ctk in the nucleus of myeloid cells (Yamaguchi et al. 2001) and its enrichment in the nucleus of epitheliotropic intestinal T-cell lymphoma (Chen et al. 2016; Tan et al. 2011, 2013). Further studies are needed to clearly define functions of this N-terminal segment of (p56Ctk).
Functions of Ctk
Phenotypes of Ctk-Deficient Mice
Three groups of researchers reported the generation of Ctk knock-out mice and their lack of overt phenotypic defects (Hamaguchi et al. 1996; Lee et al. 2006; Samokhvalov et al. 1997). Further investigation revealed subtle differences in immune responses exhibited by wild type and Ctk−/− mice. For example, upon interleukin-7 stimulation, cultured bone marrow cells derived from Ctk−/− mice but not those derived from wild type mice exhibited increased proliferation to form pre-B cells (Lee et al. 2006), suggesting that Ctk inhibits proliferation of pre-B cells. In addition, cultured T-cells from Ctk −/− mice exhibited impaired interferon γ production. Taken together, these findings indicate that Ctk plays a unique role in immune function. Furthermore, the milder-than-expected phenotypes of Ctk −/− mice suggest that cells in the Ctk −/− mice rewired their signaling pathways to allow Csk to compensate for the lack of Ctk. Conditional knockout enabling inducible inactivation of Ctk gene in specific tissues of adult mice is a more suitable approach to define the physiological function of Ctk.
Mechanism of Inhibition of Src-Family Kinases (SFKs)
Since aberrant activation of SFKs contributes to formation of cancer, in normal cells, they are kept at the inactive conformation mainly by Csk and Ctk. As shown in Fig. 1, SFKs in the inactive conformation are phosphorylated at the C-terminal tail tyrosine by Csk or Ctk. Two intramolecular inhibitory interactions are required to keep the SFKs in the inactive state: (i) binding of the phosphorylated C-terminal tail tyrosine to the SH2 domain and (ii) binding of the SH2-kinase linker to the SH3 domain. These interactions stabilize the kinase domain in the catalytically inactive state. Disruption of one or both inhibitory interactions activates SFKs. The active SFKs then undergo autophosphorylation at a conserved tyrosine (e.g., Tyr-416 of c-Src) in the kinase domain and/or dephosphorylation of the phosphorylated C-terminal tail tyrosine by a phosphatase. Upon autophosphorylation, SFKs prefer to adopt the active conformation (reviewed in Chong et al. 2005b).
How might Ctk bind SFKs and suppress their activity? Relevant to this question, Ctk was found to bind active forms of SFKs while Csk exhibited binding with much lower affinity or no binding at all (Chong et al. 2006; Levinson et al. 2008). This suggests that in addition to the interactions of basic residues in the αD helices and αF-αG loops of Ctk with acidic and hydrophobic residues in the αI helix of SFKs, additional motifs exist in both Ctk and SFKs to mediate the tight binding to form the stable Ctk/SFK complexes. Furthermore, it is logical to predict that the interactions of Ctk with SFKs via these additional motifs underpin the ability of Ctk to suppress SFK activity by the noncatalytic inhibitory mechanism. Elucidation of the structural basis of the Ctk noncatalytic inhibitory mechanism awaits definition of these unknown additional interaction motifs in Ctk and SFKs. The best approach to define these motifs is to determine the crystal structures of the Ctk/SFK complexes.
SFK-Independent Functions of Ctk
SFKs are not the only physiological substrates of Ctk. There is a growing body of evidence suggesting that Ctk is capable of phosphorylating other cellular proteins to perform SFK-independent functions. For example, Ctk expression causes activation of Erk1/2 MAP kinase signaling pathway in SFY −/− cells which lack SFKs (Zagozdzon et al. 2006), suggesting that the activation was governed by an SFK-independent mechanism. Relevant to these findings, Ctk overexpression could induce phosphorylation of an immunoglobulin superfamily protein called the tyrosine-protein phosphatase nonreceptor substrate 1 (SHPS-1) in PC12 cells (Mitsuhashi et al. 2008). The authors provided data suggesting that Ctk binds to SHPS-1 and directly phosphorylates it at Tyr-428, Tyr-452, Tyr-469, and Tyr-495. Besides PC12 cells, Ctk binding and phosphorylation of SHPS-1 was also detected in cultured vascular smooth muscle cells (Radhakrishnan et al. 2011; Shen et al. 2009). Radhakrishnan et al. demonstrated that stimulation of vascular smooth muscle cells with insulin-like growth factor-1 (IGF-1) in the presence of a high concentration (25 mM) of glucose enhanced binding of Ctk to SHPS-1 and its subsequent phosphorylation (Radhakrishnan et al. 2011). Further investigation revealed the following series of signaling events: (i) the activated IGF-1 receptor first phosphorylates Tyr-469 and Tyr-495 of SHPS-1, (ii) binding of Ctk to one of the phosphorylated tyrosines via its SH2 domain, and (iii) phosphorylation of Tyr-428 and Tyr-452 of SHPS-1 by the bound Ctk. Upon phosphorylation by Ctk, the phosphorylated SHPS-1 acts as a molecular scaffold, which directs activation of Ras and Erk1/2 MAP kinase in PC12 and vascular smooth muscle cells (Mitsuhashi et al. 2008; Radhakrishnan et al. 2011; Shen et al. 2009).
Besides SFKs and SHPS-1, Ctk may also phosphorylate other cellular proteins. Using biochemical approaches, we identified β-synuclein as a potential physiological substrate of Ctk and mapped Tyr-127 of β-synuclein as the preferential phosphorylation site (Ia et al. 2011). In the same study, we employed the arrayed positional scanning peptide libraries to define the optimal phosphorylation sequence of Ctk as E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ stands for a hydrophobic residue and x represents any amino acid residues (Ia et al. 2011). Close inspection of the SHPS-1 sequence reveals that the sequence around Tyr-452 (HTEYASI) and that around Tyr-495 (FSEYASV) conform well with the optimal phosphorylation sequence of Ctk.
Structure and Regulation of Ctk
Structure and Domain Organization of Ctk
Ctk has similar domain organization like its Csk counterpart. Namely, it has a Src-homology 3 (SH3), an SH2, and a kinase domain . In addition, the SH3 and SH2 domains are connected by the SH3-SH2 connector while the SH2 and kinase domain are connected through the SH2-kinase linker (Fig. 2). However, unlike c-Src Ctk lacks an N-terminal fatty acid acylation site, an autophosphorylation site and a C-terminal regulatory tyrosine phosphorylation site (reviewed in Chong et al. 2005a). Because of the absence of the N-terminal fatty acid acylation motif which targets cellular proteins to the plasma membrane, Ctk resides mostly in the cytosol and nucleus (reviewed in Chong et al. 2005a). It is well established that intramolecular interactions of the Csk kinase domain with the SH2 and SH3 domains play a significant role in regulating Csk kinase activity (Lin et al. 2005; Wong et al. 2004, 2005; Huang et al. 2009). Given the similarity in the domain organizations of Csk and Ctk, it is logical to predict that both the SH2 and SH3 domains are involved in regulating the kinase activity of Ctk.
Comparison of the sequences of the SH2 and the SH3 domains of Ctk and Csk reveals significant differences in the residues dictating the binding of ligands. For example, Glu-127 which dictates the binding specificity of the Csk SH2 domain is substituted by Ile-126 at the homologous position in the Ctk SH2 domain. These structural differences account for the striking difference in binding specificity of the Ctk and Csk SH2 domains (Ayrapetov et al. 2005). In this review, we discuss how these structural differences govern the differences in functions and regulation of Csk and Ctk.
Much is known about the three-dimensional structure of Csk – the structures of Csk kinase domain, full-length Csk protein, and Csk kinase domain complexed with the c-Src kinase domains have been solved (Fig. 4) (Ogawa et al. 2002, Lamers et al. 1999 and reviewed in Ia et al. 2010). In contrast, only the crystal structure of the SH2 domain of Ctk has been solved. Further investigation using biophysical approaches revealed that Ctk SH2 exists as monomers in solution (Gunn et al. 2011). Exactly how the various functional domains arrange in the three-dimensional structure of Ctk remains unclear. Owing to the similarity of organizations of the SH3, SH2, and kinase domains in the Csk and Ctk sequence, it is logical to predict that their three-dimensional structures are very similar. Unlike SFKs, the SH2 and SH3 domains of Csk are located above the minor lobe of the kinase domain with both the SH3-SH2 connector (referred to as connector) and the SH2-kinase linker sitting atop the α-helix C (Figs. 1 and 2). Such an arrangement allows both the SH3 and SH2 domains to control the conformation of the kinase domain via the connector and linker which make intimate contacts with the kinase domain (reviewed in Chong et al. 2005b, Ia et al. 2010). It is likely that a similar arrangement of the connector, linker, and the three functional domains can be found in the three-dimensional structure of Ctk. With this arrangement, binding of the cognate ligands to the SH2 and SH3 domains is expected to modulate kinase domain conformation and hence the kinase activity of Ctk.
Specificities of SH2 and SH3 Domains
Using the SMALI algorithm developed by Huang et al. (Huang et al. 2008), a number of tyrosine-phosphorylated cellular proteins were predicted to be recognized by the Ctk SH2 domain. Among them are the growth factor receptor kinases ErbB2 and c-Kit, which were previously identified to be ligands of the Ctk SH2 domain (reviewed in (Chong et al. 2005a)).
Little is known about Ctk SH3 domain binding specificity. Similar to the Csk SH3 domain, binding of the Ctk SH3 domain to its ligand is expected to govern the subcellular localization of Ctk. Identification of the ligands targeting the Ctk SH3 domain will provide further insight into the function and regulation of Ctk. Most SH3 domains bind peptide ligands with the –PxxP- motif, the two prolines in the motif interact with four conserved hydrophobic aromatic residues in the SH3 domain. In Csk, they are Tyr-18, Phe-20, Trp-47, and Tyr-64 (Fig. 2). Intriguingly, three out of four of these residues are substituted by the less hydrophobic residues (Cys-17, Asn-19, and Ala-63) in Ctk (Fig. 2). It is unclear whether the lack of hydrophobic aromatic residues at the PxxP motif-binding pocket affects the specificity of the Ctk SH3 domain’s binding to cellular proteins. In addition to binding ligand, the SH3 domain was demonstrated to mediate dimerization of Csk (Levinson et al. 2009). In contrast to Csk, Ctk exists as a monomer in solution (Chan et al. 2010), indicating that the Ctk SH3 domain does not govern dimerization of the enzyme.
Regulation of Kinase Activity of Ctk
Chan et al. (Chan et al. 2010) compared the specific enzymatic activities of recombinant Ctk and Csk and found that they exhibit similar efficiency in phosphorylating the SFK member Lyn, indicating that the basal activity of both kinases are similar. Other than this, little is known about the regulation of Ctk activity. In contrast, it is well documented that the SH2 and SH3 domains play significant roles in governing the kinase activity of Csk (reviewed in Ia et al. 2010). Owing to the high degree of sequence homology between Ctk and Csk (Figs. 2 and 4), structural comparison of both kinases allows us to make predictions of Ctk regulation.
The presence of SH2 and SH3 domains suggests that Ctk kinase activity is likely to be regulated by protein ligands that bind to these two domains (Fig. 3). The activity of many protein kinases is governed by the orientation of a conserved structural motif in the kinase domain called the α-helix C (Fig. 3) (see Ia et al. 2010 for review). Results from biochemical analysis by Mikkola and Bergman (reviewed in Chong et al. 2005b) suggest that the SH2-kinase linker interacts with the α-helix C and controls Ctk kinase activity. Their results imply that protein ligands that specifically bind to the SH2 domain of Ctk can regulate Ctk activity by modulating this interaction. Of relevance, the SH2 domain of Ctk was reported to bind to a motif containing the phosphorylated Tyr-1248 of ErbB2 (Fig. 6). It will be worthwhile to examine if and how binding of Ctk to the phosphorylated ErbB2 affects Ctk kinase activity and its efficiency in inhibiting SFKs. Unlike many other protein kinases, Ctk lacks the conserved autophosphorylation tyrosine in the activation loop of the kinase domain, suggesting that it is not regulated by tyrosine phosphorylation of the activation loop.
Regulation of Subcellular Localization of Ctk
Ctk comprises of three functional domains (SH3, SH2 domain, and kinase domains), an N-terminal motif unique to each isoform and a C-terminal tail. Unlike SFKs, Ctk lacks the N-terminal fatty acid acylation domain. For this reason, Ctk resides predominantly in the cytosol in a number of cell types. Apart from directly binding to SFKs to form stable protein complexes (Chong et al. 2006), Ctk interacts with three transmembrane receptor tyrosine kinases including ErbB2, c-kit, and TrkA; the focal adhesion kinase-related kinase Pyk2/RAFTK; and the scaffolding protein paxillin (reviewed in Chong et al. 2005a, b). Since SFKs are localized to the plasma membrane, endosomes, and perinuclear regions (Seong et al. 2009), Ctk needs to be recruited from the cytosol to these organelles to inhibit SFKs. Both the SH2 and SH3 domains are involved in targeting Ctk to specific subcellular compartments to perform its physiological functions.
The SH2 domain of Ctk mediates its binding to a number of transmembrane receptor tyrosine kinases and intracellular kinases. Upon stimulation of the EGF-receptor family tyrosine kinase ErbB2 in breast cancer cell lines by heregulin, Ctk binds to ErbB2. The binding is mediated by the Ctk SH2 domain and the phosphorylated Tyr-1248 residue of activated ErbB2 (Fig. 6) (reviewed in Cheng et al. 2006, Chong et al. 2005a). As a consequence of Ctk-ErbB2 binding, Ctk suppresses SFKs activated by ErbB2 and breast cancer cell growth. In addition to ErbB2, Ctk also employs its SH2 domain to bind to several other transmembrane receptor tyrosine kinases. In the rat pheochromocytoma PC12 cell line, Ctk SH2 domain binds to the nerve growth factor (NGF)-receptor TrkA (Fig. 6). The binding was implicated in modulating NGF-induced differentiation of the rat pheochromocytoma PC12 cell lines (reviewed in Cheng et al. 2006, Chong et al. 2005a). Ctk SH2 domain also binds to stem cell factor (SCF) receptor c-Kit in hematopoietic cells. The binding was thought to be essential for SFK inhibition (reviewed in Cheng et al. 2006, Chong et al. 2005a). Ctk SH2 domain also mediates its binding to the focal adhesion kinase-related kinase Pyk2/RAFTK in breast cancer cells stimulated with heregulin (reviewed in Cheng et al. 2006, Chong et al. 2005a). The binding reduces tyrosine phosphorylation of Pyk2/RAFTK and of the focal adhesion-associated protein paxillin.
Although ligands of the Ctk SH3 domain have yet to be identified, evidence provided by Hirao et al. suggests that the Ctk SH3 domain can interact with an unknown adaptor protein located on the plasma membrane in megakaryocytic Dami cells. The binding recruits Ctk to the plasma membrane. The recruitment is associated with inhibition of the SFK member Lyn and may contribute to suppression of fibronectin-stimulated cell spreading (reviewed in Chong et al. 2005a).
Ctk as a Potential Tumor Suppressor
Overactivation and overexpression of SFKs are known to contribute to many forms of cancers such as breast and colon carcinoma, and leukemia (Cheng et al. 2006; O’Hare et al. 2008 and reference quoted therein). Being a major endogenous inhibitor of SFKs, Ctk is a potential tumor suppressor. Of relevance, Ctk expression is suppressed in colon carcinoma cell lines and biopsies from patients suffering from colon cancer. Furthermore, expression of recombinant Ctk in colon cancer cells suppresses SFK activity, anchorage-independent growth, and cell invasion (Zhu et al. 2008). In addition, overexpression of Ctk was found to suppress growth and proliferation of human breast carcinoma cells (Kim et al. 2002; Zrihan-Licht et al. 1997; Dokmanovic et al. 2014 and reviewed in Cheng et al. 2006; Chong et al. 2005a).
Besides phosphorylating and inhibiting SFKs, Ctk can potentially exert its tumor suppressive action by phosphorylating other cellular substrates such as SHPS-1. The quantitative phosphoproteomics approach is one avenue to define the tumor suppressive signaling mechanism of Ctk in cancer cells. This approach entails induced expression of recombinant Ctk in the Ctk-deficient cancer cells such as colorectal carcinoma cells (Zhu et al. 2008). Comparison of the phosphoproteomes of the cancer cells with and without expression of Ctk will unveil cellular proteins of which the phosphorylation levels are perturbed by induced expression of Ctk. Further investigation to define how Ctk modulates the phosphorylation and functions of these cellular proteins will shed light on the tumor suppressive signaling mechanism of Ctk.
Is Ctk Expression Downregulated by Epigenetic Silencing in Cancer Cells?
Reports by several groups of investigators confirm that expression of Ctk is suppression in brain tumors and colorectal cancer cell lines and tissue biopsies (Zhu et al. 2008; Laffaire et al. 2011; Kim et al. 2004). However, exactly how Ctk expression is suppressed in cancer cells remains unclear. Using an unbiased genome-wide methylation detection technique, hypermethylation of the Ctk promoter was observed in low-grade glioma (in 93% of tumor samples) which is consistent with previous reports demonstrating significant downregulation of Ctk in brain tumors (Laffaire et al. 2011). In agreement to our observation of reduced expression of Ctk in colorectal cancer cell lines and biopsies (Zhu et al. 2008), Ctk promoter was found to be significantly hypermethylated in the CpG island methylator phenotype-positive (CIMP-positive) colorectal tumors (Hinoue et al. 2012). Further work is needed to unequivocally establish the role of epigenetic silencing in suppression of Ctk expression in cancer cells. Experimental approaches to define the mechanism of suppression of Ctk expression include (i) bisulfite sequencing of the promoter of Ctk gene of colorectal cancer and brain tumor cells to define the hypermethylated CpG sites, (ii) the use of epigenetic silencing inhibitors such as 5-Azacytidine to ascertain whether inhibition of DNA methyltransferases can restore expression of Ctk in these cancer cells, and (iii) investigation of the effects of restoration of expression of Ctk on the oncogenic phenotypes such as cell proliferation and anchorage-independent growth of the cancer cells.
Studies of the phenotypes of Ctk −/− mice and biochemical studies of Ctk functions in cancer cell lines demonstrated that Ctk plays unique roles in modulating immune cell signaling and functions as a tumor suppressor. Presumably, these functions of Ctk are attributed to its ability to suppress the activity of SFKs. In addition to hematopoietic cells, neurons also express high level of Ctk. Little is known about the function of Ctk in neurons. Future investigation should focus on elucidating the mechanism of regulation and functions of Ctk in neurons. Even though Ctk is a major endogenous inhibitor of SFKs, little is known about the structure, regulation, and the structural basis of its inhibition of SFKs. There are two important outstanding questions concerning the structure and function of Ctk: What is the structural basis of the noncatalytic inhibitory mechanism employed by Ctk to inhibit SFK activity? What are the other physiological substrates of Ctk?
Deciphering the structural basis of Ctk inhibition of SFKs by the noncatalytic inhibitory mechanism requires mapping of the SFK-binding determinants in Ctk and the Ctk-binding determinants in SFK. We demonstrated in our previous study that these determinants reside in the kinase domain of both Ctk and SFKs (Chong et al. 2006). As shown in Fig. 5, similar to Csk, the conserved basic residues in the αD-helix and αF-αG loop of Ctk are likely SFK-binding determinants. However, the binding of Ctk to SFKs is much tighter than that of Csk to SFKs (Chong et al. 2006). It is logical to predict that in addition to these determinants in the αD-helix and αF-αG loop, determinants residing in other regions of Ctk kinase domain are required for its tight binding to SFKs. Further biochemical analyses are needed to map these determinants. Finally, determination of the three-dimensional structures of Ctk and Ctk/c-Src complex will provide valuable insights into the structural basis of this noncatalytic inhibitory mechanism of Ctk.
There is no doubt that small molecule inhibitors specifically inhibiting SFKs are needed for development as therapeutics for cancer treatment. A number of inhibitors targeting the ATP-binding pocket of the active SFKs have been developed for clinical use or for preclinical studies. Among them, Dasatinib, AZD0530, and SKI-606 are currently in clinical trials for the treatment of different forms of cancer. The use of Dasatinib for the treatment of drug-resistant chronic myelogenous leukemia has been approved. Owing to the ability of these small molecule compounds to inhibit other protein kinases and possibly some nonprotein kinase enzymes, use of these compounds for the treatment of cancer can cause significant side effects in patients receiving the treatment. Thus, the second generation chemical inhibitors which target SFKs with exquisite selectivity are urgently needed. Since Ctk can employ the noncatalytic inhibitory mechanism to specifically suppress the activity of SFKs adopting the active conformations. Future investigation to define the structural basis of the noncatalytic inhibitory mechanism of Ctk will benefit the development of this new generation of inhibitors.
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