Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Csk

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_264

Synonyms

Historical Background

The activity of the  Src family tyrosine kinases (SFKs), known as representative proto-oncogene products, is negatively regulated by the phosphorylation at their C-terminal regulatory tyrosine (Brown and Cooper 1996; Cooper et al. 1986). The protein tyrosine kinase Csk was identified as a specific kinase that directs the negative regulatory sites of SFKs (Nada et al. 1991; Okada and Nakagawa 1988). Analysis of Csk-deficient mice and cells provided evidence that Csk functions as an indispensable negative regulator of SFKs (Nada et al. 1991, 1993). The molecular basis of Csk-SFK interaction is recently verified by the crystal structure of Csk/c-Src complex (Levinson et al. 2008). Csk is expressed ubiquitously, but is highly concentrated in developing nervous system and the immune system (Okada et al. 1991). Csk is highly conserved in animal kingdom from the unicellular choanoflagellate to human in parallel with SFKs (Segawa et al. 2006). Csk is a cytosolic protein, consisting of the kinase domain and the conserved domains responsible for the protein-protein interaction (Ogawa et al. 2002) (Fig. 1). Several Csk-binding proteins, for example, Cbp/PAG1 and paxillin, were identified as specific scaffolds that recruit Csk to the sites where SFK is activated (Kawabuchi et al. 2000; Oneyama et al. 2008a; Sabe et al. 1994). The Csk-mediated inhibition of SFKs is crucial for suppressing oncogenic ability of SFKs (Oneyama et al. 2008b).
Csk, Fig. 1

Structural features of SFKs and Csk. (a) Domain organization of Csk. SH3; Src homology 3 domain, SH2; Src homology 2 domain. The ligands of SH3 and SH2 domains, PPII and pY, respectively are indicated. PPII; the polyproline type II helical structures, pY; the phosphotyrosine-containing protein ligands. The kinase domain consists of N-lobe and C-lobe. Active site and activation loop are indicated. (b) Schematic representation of the crystal structure of Csk. The binding pockets of SH3 and SH2 domains of Csk are oriented outward enabling the intermolecular interactions. Csk adopts active and inactive conformations. The 60° rotation of the SH2 domain, associated with the active-inactive form transition, is indicated by an arrow. (c) Domain organization of SFKs. SFKs has fatty acyl moieties (myristate and/or palmitate) and the non-conserved unique domains (SH4) in the N-terminal region. The autophosphorylation site (Y418) in the activation loop and the C-terminal negative regulatory site (Y529) are indicated. (d) Schematic representation of the inactive and active structures of SFKs based on the crystal structures of c-Src and Hck. The intramolecular interactions between pY529 and SH2 domain and between SH2-kinase linker and SH3 domain stabilize the inactive conformation. Dephosphorylation of pY529 unlocks the inactive conformation and the trans-autophosphorylation at Y418 makes the enzyme fully active

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

Csk is predominantly present in cytosol due to the lack of fatty acyl modification, while its substrate SFKs are anchored to the membrane via the N-terminal myristate and palmitate moieties. Thus, the translocation of Csk to the membrane, where SFKs are activated, is one of the critical steps of Csk regulation (Howell and Cooper 1994). So far several scaffolding proteins have been shown to serve as anchors of Csk to the membrane. A well-characterized example is Cbp/PAG1 (Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched membrane) that is a transmembrane protein having both myristoyl and palmitoyl modifications like SFKs. Cbp/PAG1 is highly concentrated in the cholesterol-enriched membrane microdomain “lipid rafts” and serves an excellent substrate of SFKs. Upon phosphorylation of Y314 of Cbp/PAG1 by the activated SFKs, pY314 binds to the SH2 domain of Csk, and this in turn recruits Csk to the plasma membrane. Csk on the plasma membrane then efficiently inactivates SFKs that are also recruited to Cbp/PAG1. This negative feedback loop is crucial in preventing tumorigenesis and in controlling the cell signaling evoked by the activation of growth factor receptors (Fig. 2). Other Csk-binding proteins, such as paxillin (Sabe et al. 1994) and caveorin-1 (Cao et al. 2002), are also phosphorylated by SFKs and are involved in the negative regulation of SFKs by recruiting Csk to the sites where SFKs are activated in a manner similar to Cbp/PAG1.
Csk, Fig. 2

Negative regulatory loop of SFK via Cbp/PAG1 on lipid rafts. When SFKs are activated in response to cell stimulation, they phosphorylate Cbp/PAG1 residing in lipid rafts to create a binding site of Csk (pY314) and their own binding sites (pY381/409). Inactive Csk in cytosol is then recruited to the phosphorylated Cbp/PAG1, and the activated Csk efficiently phosphorylates Y529 of SFKs that are also recruited to Cbp/PAG1. The phosphorylated SFKs are released from Cbp/PAG1 by restoring the intramolecular interaction between SH2 domain and pY529

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.

Summary

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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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Oncogene Research, Research Institute for Microbial DiseasesOsaka UniversitySuita, OsakaJapan