Regulation of the Src Family Kinases by Csk Masato Okada
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Int. J. Biol. Sci. 2012, 8 1385 Ivyspring International Publisher International Journal of Biological Sciences 2012; 8(10):1385-1397. doi: 10.7150/ijbs.5141 Review Regulation of the Src Family Kinases by Csk Masato Okada Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, JAPAN. Corresponding author: TEL: 81-6-6879-8297 FAX: 81-6-6879-8298, E-mail: [email protected]. © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2012.08.31; Accepted: 2012.10.01; Published: 2012.11.01 Abstract The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of the Src family tyrosine kinases (SFKs) by specifically phosphorylating the negative regulatory site of SFKs, thereby suppressing their oncogenic potential. Csk is primarily regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG1. The binding of scaffolds to the SH2 domain can also upregulate Csk kinase activity. These regulatory features have been elucidated by analyses of Csk structure at the atomic levels. Although Csk itself may not be mutated in human cancers, perturbation of the regulatory system consisting of Csk, Cbp/PAG1, or other scaffolds, and certain tyrosine phosphatases may explain the upregulation of SFKs frequently observed in human cancers. This review focuses on the molecular bases for the function, structure, and regulation of Csk as a unique regulatory tyrosine kinase for SFKs. Key words: Csk, Src family, tyrosine kinases The Src family tyrosine kinases The Src family tyrosine kinases (SFKs) are the major non-receptor tyrosine kinases expressed in Tyrosine kinases, which exist only in animals, multiple types of animal cells [3]. The SFKs include play important roles in controlling animal-specific eight members, c-Src, c-Yes, Fyn, c-Fgr, Lyn, Hck, Lck, cellular functions such as rapid cell–cell communica- Blk. They are activated in concert with the activation tion via the plasma membrane [1, 2]. The most critical of transmembrane receptors, such as EGF receptor, T feature of tyrosine kinases is the strict regulation of cell receptor, and integrins. The SFKs regulate a wide their functions. Due to these enzyme’s importance in range of cellular events, including cell growth, divi- controlling cellular functions, dysregulation of tyro- sion, differentiation, survival, and programmed sine kinases can play a causative role in various dis- death, as well as specialized functions such as im- eases, especially cancers. Tyrosine kinases are classi- mune responses, cell adhesion, cell movement, and fied into two major subgroups: receptor and endocytosis [4, 5]. Several of the SFKs, c-Src, c-Yes, non-receptor. The receptor tyrosine kinases are acti- and c-Fgr, were originally discovered as prototypes of vated and auto-phosphorylated upon directly receiv- avian retroviral oncogene products (v-Src, v-Yes and ing extra-cellular cues, creating an intracellular plat- v-Fgr) [6-11]. Dysregulation of SFKs has been impli- form for specific adaptors and effectors that activate cated in the etiology of various human diseases such downstream signaling pathways. The cytoplasmic as cancers, although somatic mutation in the SFK non-receptor tyrosine kinases are also activated in genes has not been detected [12, 13]. There observa- response to extra-cellular cues via physical and func- tions underscore the important roles played by SFKs. tional interactions with particular transmembrane However, the molecular mechanisms underlying the receptors, and subsequently activate specific down- stream pathways to control cell fate. http://www.biolsci.org Int. J. Biol. Sci. 2012, 8 1386 causes of SFK dysregulation in the diseases remain SFKs (Fig.1B). Palmitoylation has been implicated in unclear. the stable localization of SFKs in membrane micro- The overall sequences of SFKs, with the excep- domains called “lipid rafts” [14]. All SFKs share the tion of the N-terminal ~50 residues, is highly con- Src homology 3 (SH3) and SH2 domains, the kinase served among the family members. The primary domain, and the C-terminal regulatory tail. These structure can be divided into five functional domains structural similarities allow for common mechanisms (Fig.1A). The N-terminal domain contains signals for of regulation: the phosphorylation of the conserved lipid modification: myristoylation (in all SFKs) and tyrosine residues (Tyr-416 and Tyr-527) and the in- palmitoylation (in all but c-Src and Blk) signals, both tramolecular interactions among the domains are of which are required for membrane association of crucial for the regulation of SFKs (Fig.1B). Fig.1. The Src family kinases (SFKs). (A) Domain organization of SFKs. (B) Amino acid alignments of N-terminal unique domain, activation loop and C-terminal regulatory tail in SFKs. In 1985, Courtneidge first recognized that c-Src is constitutively active. By contrast, Tyr-416 in the cata- activated by dephosphorylation [15]. Cooper and lytic domain is highly phosphorylated in v-Src and a King subsequently identified the conserved Tyr-527 c-Src mutant bearing a Tyr-527 to Phe substitution (in chicken c-Src) in the C-terminal tail as the site of (c-SrcF527) [17, 18], suggesting that these two tyrosine inhibitory phosphorylation [16]. These findings phosphorylation sites are involved in a reciprocal demonstrate that SFK is normally present in its inac- regulatory mechanism. The detailed mechanism of tive form, in which Tyr-527 is phosphorylated. This is SFK regulation was later resolved at the atomic level consistent with the fact that the oncogenic v-Src, by analyses of the crystal structures of the inactive which lacks the C-terminal tail containing Tyr-527, is and active forms of c-Src [19-21] (Fig.2A). In resting http://www.biolsci.org Int. J. Biol. Sci. 2012, 8 1387 cells, SFKs are mostly phosphorylated at Tyr-527, and The regulation of SFKs by intramolecular inter- adopt the inactive conformation, which is stabilized actions suggests that SFKs can also be regulated by by two intramolecular interactions: 1) binding of interaction with molecules that compete with the SFK phosphorylated Tyr-527 to its own SH2 domain, and domains. Indeed, SFKs bind to various tyro- 2) binding of the SH2-kinase linker to the SH3 do- sine-phosphorylated proteins by recognizing specific main. These intramolecular interactions affect the phosphopeptide sequences via the SH2 domain [23]. configuration of the catalytic pocket. In particular, the For example, c-Src binds to the phosphorylated forms hydrogen bond between Glu-310 in the αC-helix and of Cas, FAK, and paxillin, as well as growth factor Lys-295, required for Mg-ATP binding, is disrupted in receptors such as EGFR, CSF-1 and PDGF, resulting in the inactive conformation. The dephosphorylation of activation of c-Src [5, 24-26]. Activated c-Src can fur- Tyr-527 releases the ‘lock’ by the SH2 domain and ther phosphorylate these interacting proteins to create causes dramatic conformational change in the kinase new binding sites for other adaptors and effectors, domain, resulting in formation of a hydrogen bond which in turn allows amplification of signals. In addi- between Glu-310 and Lys-295 in the catalytic domain tion, the c-Src SH3 domain binds to various signaling (Fig 2A). This active enzyme can now catalyze the proteins that contain proline-rich motifs [27], such as intermolecular auto-phosphorylation of Tyr-416 in the Shc, PI3K, Cas, Tks5, and Arhgef5 [28-30]. Interactions activation loop. This auto-phosphorylation locks the with these proteins disrupt the stabilized inactive catalytic domain into the active conformation and conformations of SFKs and promote the phosphory- facilitates access of substrates to the active site [22]. lation of these proteins by SFKs. Fig.2. Mechanism of SFK activation. (A) Schematic models of inactive and active forms of c-Src (cited from [21]). (B) A predicted mechanism of SFK activation. http://www.biolsci.org Int. J. Biol. Sci. 2012, 8 1388 Although the molecular mechanisms of SFK ac- model system [37]. Furthermore, we and another tivation may vary depending upon cell types and ex- group showed that Csk phosphorylates the conserved tracellular cues, it is now believed that in general, full negative regulatory sites in other SFKs [36, 38]. Sabe et activation of SFK is achieved in the following order al. showed that when Csk is overexpressed, it inhibits (Fig.2B): 1) the activated receptors, adaptors, or ef- transformation of fibroblasts by the combined actions fectors interact with the SH2/3 domains of inactive of c-Src and the oncoprotein v-Crk [39, 40]. This ob- SFKs to open up the closed conformation, and harbor servation suggests that Csk has the potential to serve SFKs at appropriate intracellular locations such as as a repressor of c-Src in vivo as well as in vitro. focal adhesions; and 2) tyrosine phosphatases dephosphorylate the exposed pTyr-527 to stabilize the active conformation; and 3) the activated SFKs un- dergo intermolecular auto-phosphorylation on Tyr-416 to lock the catalytic pocket into the fully ac- tive conformation. The fully activated SFKs then phosphorylate substrate proteins, such as FAK and Cas, many of which can also create binding sites for SFKs; this initiates the positive-feedback loop of SFK activation. When the cell response is terminated, ac- tivated SFKs are rapidly degraded via the ubiqui- tin-proteasome pathway [31] or inactivated by phos- phorylation at Tyr-527. Thus, in order to guarantee rapid cell response to extracellular cues, most of SFKs in the resting state should be inactivated by phos- phorylation at Tyr-527.