Regulation of the SRC family kinases by Csk

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.

Keywords: Csk; Src family; tyrosine kinases.

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.

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.

Fig 3

Discovery of Csk. (A) Sample of each purification step of Csk was analysed by SDS-PAGE and staining with silver. Lane 1, Nonidet P-40 extract; lane 2, DEAE-cellulose column chromatography; lane 3, poly(Glu, Tyr) Sepharose CL-4B: lane 4, Mono Q; lane 5, Sephacryl S200HR; lane 6, Mono S. (B) Phosphorylaion of Src by Csk was determined by incubating immunopurified SrcWT (lanes 1 and 2), SrcKD (lanes 3 and 4) and SrcKDF527 (lanes 5 and 6) with or without Csk. The reaction products were analyzed by SDS-PAGE and autoradiography.

Fig 4

Function of Csk. (A: upper) Histological appearances of E8.0 embryos. Transverse sections through the trunk region of wild-type (+/+) and Csk KO (-/-) embryos, respectively. The neural lube began to close in the wild-type embryo, but not in the homozygous mutant embryos. (A: lower left) Activation of SFKs in Csk KO embryos. Src, Fyn and Lyn were immunopurifed from the indicated genotypes of embryos and incubated with acid-treated enolase and [γ-³²P]ATP, followed by SDS-PAGE. The Incorporation of ³²P to enolase was quantitated. (A: lower wright) Tyrosine-phosphorylated proteins in the indicated genotypes of embryos were analyzed by Western blotting with anti-phosphotyrosine antibody. (B) Csk can phosphorylate the C-terminal regulatory sites of all the members of SFKs to repress their activities.

Fig 5

Evolution of SFKs and Csk. Phylogenetic tree of Src, Csk, and related genes by using the maximum likelihood method. The reliability index (1) and bootstrap probability (2) for each branch are indicated before and after the slash, respectively.

Fig 6

Structure of Csk. (A) Domain organizations of Csk and SFKs. An amino acid alignment of the activation loop of Csk and SFKs is also shown. (B) Ribon diagram of a representative Csk structure (active molecule) in the crystal.

Fig 7

Regulation of Csk. (A) Superimposed model of the six Csk molecules in an asymmetric unit. Active molecules are shown in green and inactive molecules are in blue. (B) Schematic models of active and inactive Csk structures. (B: boxes) Arrangements of the catalytically important residues in active and inactive Csk. The salt bridge between Lys-222 and Glu-236 is indicated by dotted lines.

Fig 8

Regulation of Csk via Cbp in lipid rafts. A schematic model of the roles of Cbp/Csk and lipid rafts in regulating the function of SFKs. When Cbp is phosphorylated by active SFKs, Csk is recruited to lipid rafts via binding to Cbp at pY314 and phosphorylates Y527 to inactivate the catalytic activity of SFKs. The inactivated SFKs then relocate to non-raft compartments.

Fig 9

Function of Csk as a tumor suppressor. (A) Effect of Csk overexpression on tumor growth. Human colon cancer cell line HT29, HT29 expressing wild-type Csk (HT29/Csk) and HT29 expressing kinase-negative Csk (HT29/CskKN) were injected s.c. into nude mice. Tumor volume (cm³) obtained from four mice is plotted versus days after inoculation. Excised tumors are shown in lower panel. (B) Tumor incidence in wild-type and heterozygous for Csk (csk^+/-) and p53 (p53^+/-) after the sequential treatment of skin with dimethyl benzanthracene (DMBA) and phorbol myristate acetate (TPA).