Structure of Csk
Csk is a non-receptor type of protein tyrosine kinase with a molecular mass of 50 kDa. It contains a Src homology 3 (SH3) and a Src homology 2 (SH2) domains in its N-terminal half and a kinase domain in its C-terminus. This primary structural arrangement of functional domains is similar to that of SFKs, but Csk lacks the N-terminal fatty acylation sites, the autophosphorylation site in the activation loop, and the C-terminal negative regulatory sites, all of which are crucial for regulating SFK activity (Fig. 1). The lack of autophosphorylation is a unique feature as a protein tyrosine kinase. The crystal structures of inactive and active forms of Src reveal the regulatory mechanism of SFKs (Cowan-Jacob et al. 2005; Xu et al. 1997). Upon phosphorylation at the C-terminal tyrosine, SFKs adopt the inactive conformation stabilized by two intramolecular inhibitory interactions: (1) binding of the C-terminal phosphotyrosine to the SH2 domain and (2) binding of the SH2-kinase linker to the SH3 domain. The dephosphorylation of the C-terminal tyrosine results in an open structure where the kinase domain adopts an active conformation (Fig. 1).
The crystal structure of Csk reveals significantly different dispositions of the functional domains from those of SFKs (Ogawa et al. 2002), indicating that Csk is regulated differently to SFKs (Fig. 1). The most intriguing difference in the domain organization between Csk and SFKs is that the binding pockets of SH3 and SH2 domains of Csk are oriented outward, enabling the intermolecular interactions. The crystal structure of Csk further predicts that Csk can adopt active and inactive conformations. It is suggested that the kinase domain of Csk is intrinsically inactive, but the direct interaction with the SH2 domain induces conformational change, resulting in an upregulation of the kinase activity (Wong et al. 2005).
Function of Csk
Accumulated evidence shows that SFKs are the major physiological substrates of Csk. Csk-deficient mice exhibit early embryonic lethality, accompanied by a constitutive activation of c-Src, Fyn, and Lyn (Nada et al. 1993). Conditional mutagenesis of the csk gene in specific tissues causes severe defects that are associated with constitutive activation of SFKs. Loss of Csk induces dysfunction in acute inflammatory responses (Thomas et al. 2004) and T-cell development (Schmedt et al. 1998), hyperplasia of the epidermis (Yagi et al. 2007), and defects in cell adhesion and migration (Nada et al. 1994). Even in invertebrates, such as Drosophila and C. elegans, the loss of Csk leads to constitutive activation of SFKs, causing hyperproliferation and defective cytoskeletal function, respectively (Read et al. 2004; Takata et al. 2009). These findings indicate that Csk is an indispensable regulator of SFKs.
As a protein tyrosine kinase, Csk has an exceptionally high specificity for the C-terminal regulatory tyrosine (Y527) of SFKs. The surrounding sequence of the regulatory site of SFKs (QYQ) is unique and well conserved among SFKs, but biochemical and structural studies reveal that a region (aa 504–525) located distantly from Y527 is rather crucial for specific recognition by Csk (Lee et al. 2003, 2006; Levinson et al. 2008). In addition to SFKs, several signaling proteins have been reported to serves as substrates of Csk. Those include paxillin (Sabe et al. 1994), P2X3 receptor (D’Arco et al. 2009), c-Jun (Zhu et al. 2006), and Lats (Stewart et al. 2003). However, the physiological relevance of the phosphorylation of these proteins still remains unclear.
Regulation of Csk
The binding of scaffolds to the SH2 domain of Csk can also activate the enzyme activity of Csk. The occupation of the SH2 domain of Csk by phosphorylated Cbp affects conformation of the catalytic domain, thereby increasing the activity toward SFKs (Takeuchi et al. 1993; Wong et al. 2004, 2005). Thus, it is likely that the scaffold proteins positively regulate Csk functions not only by recruiting Csk to the membrane but also by directly activating Csk.
It is also reported that the activity of Csk can be regulated by the oxidation state of the disulfide bond in the SH2 domain, suggesting the regulation mechanism by the redox state (Mills et al. 2007). Furthermore, there is a report indicating that Csk is phosphorylated by PKA at S364, resulting in an increase in kinase activity (Yaqub et al. 2003). However, their physiological relevance has not yet been addressed. Although the expression of Csk is substantially high in the developing nervous system and lymphoid cells, the mechanisms underlying the regulation of Csk at the expression levels are thoroughly unknown.
Csk in Diseases
Since Csk has a tumor-suppressive function by inhibiting oncogenic activity of SFKs, it is reasonable that Csk is involved in human cancer. Although there are some reports suggesting that Csk is downregulated in some cancers (Masaki et al. 1999), it seems that Csk downregulation is rather rare and it is more likely that Csk is expressed in various cancer cells at a comparable level as a housekeeping protein. In contrast, it is clear that the expression of Cbp/PAG1 is appreciably downregulated in a variety of cancer cells (Oneyama et al. 2008a), potentially via the epigenetic mechanism (Suzuki et al. 2011). The downregulation of Cbp/PAG1 may interfere with the translocation of Csk to the membrane, thereby upregulating SFK functions. This mechanism may account for the upregulation of SFKs in some cancer cells. Thus, the further analysis of Cbp−/PAG1-mediated regulatory system would provide new opportunities for therapeutic intervention in cancer.
The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of SFKs, by specifically phosphorylating the negative regulatory site of SFKs to suppress their oncogenic potential. Csk is mainly regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG. The binding of scaffolds to the SH2 domain can also upregulate the kinase activity. These regulatory features are mostly clarified by the analysis of Csk structure at atomic levels. Although Csk itself is not directly relevant to human cancer, the perturbation of the regulation system of SFKs, which consists of Csk, Cbp/PAG1, or other scaffolds, and some tyrosine phosphatases, would be attributed to the upregulation of SFKs which is frequently observed in human cancers.
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