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  • The crystal structure of full

    2019-09-10

    The crystal structure of full-length CHK is still unsolved. However, the close homology between CHK and Csk suggests that these kinases share a similar structure. Therefore, the engagement of the CHK SH2–kinase linker with the αC-helix could control the activity of the CHK kinase domain. Sequence alignment of the CHK and Csk SH2–kinase linkers supports this hypothesis (Fig. 2A). CHK has a Leu223 within the SH2–kinase linker in a position corresponding to Csk Phe183. This conserved hydrophobicity in the CHK SH2–kinase linker favors the possibility that CHK Leu223 is essential for catalytic activity of CHK, as Phe183 is for Csk. As suggested, smad inhibitor of the side chain of Leu223 decreased the catalytic activity of CHK, whereas activity could be restored by mutating Leu223 to Phe. Together these results indicate that Leu223 is necessary for the catalytic activity of the CHK kinase domain. The most probable explanation for the obtained results is that Leu223 makes a hydrophobic contact with the αC-helix and controls the orientation of the catalytically critical Glu276 (Csk Glu236), in a manner analogous to Csk Phe183. Previous studies have indicated that mutation of the SH3–SH2 linker residues also decrease the catalytic activity of Csk [9], [17]. The SH3–SH2 linker residue Ser78 was mutated in both studies. Because Ser78 makes contact with the SH2–kinase linker (Fig. 1B), it is possible, that the SH3–SH2 linker mutation results in weaker contact between linkers. Presumably a weak contact between the SH3–SH2– and SH2–kinase linkers allows a higher degree of movement of the αBC-helix on top of the N-terminal lobe of the kinase domain. As a result, catalytic activity decreases because Phe183 in the αBC-helix cannot contact the αC-helix to support the catalytically active conformation of the αC-helix.
    Acknowledgements
    Introduction Protein–tyrosine kinases are found in all multicellular eukaryotic organisms and play important roles in a variety of intracellular signal-transduction pathways. Receptor-type tyrosine kinases, a subclass of transmembrane-spanning receptors, transmit signals across the plasma membrane from extracellular milieus to the inside of cells. However, the function of non-receptor-type tyrosine kinases depends upon their intracellular localizations [1]. The Csk homologous kinase Chk is a second member of the Csk family of non-receptor-type tyrosine kinases [2], [3], [4], [5], [6], [7], [8]. Chk is restrictedly expressed in hematopoietic and neuronal cells, whereas Csk is ubiquitously expressed [4], [5], [9], [10], [11]. Chk is composed of (i) the N-terminal unique domain, (ii) the Src homology 3 (SH3) domain, which can bind to specific proline-rich sequences, (iii) the SH2 domain, which can bind to specific sites of tyrosine phosphorylation, and (iv) the protein–tyrosine kinase domain. It is of interest to note that Chk lacks the consensus tyrosine phosphorylation and myristoylation sites found in the Src-family of non-receptor-type tyrosine kinases. Like Csk, Chk suppresses the activity of Src-family kinases by phosphorylating their C-terminal negative regulatory tyrosine residues. Although Csk negatively regulates the kinase activities of all members of Src-family kinases in vivo [12], Chk selectively suppresses the kinase activity of the Src-family kinase Lyn but not c-Src in platelets and megakaryocytic Dami cells [11], [13]. Moreover, Chk is found to bind to several tyrosine-phosphorylated growth factor-receptor tyrosine kinases, c-Kit, TrkA, and ErbB-2, via the SH2 domain of Chk [14], [15], [16]. We recently showed that Chk is localized to the nucleus as well as the cytoplasm in myeloid cells, and that overexpression of Chk in myeloid cells brings about growth retardation and multinucleation. Ectopic expression of Chk in COS-1 cells is also distributed to the nucleus and the cytoplasm leading to inhibition of cell proliferation [17]. In addition, expression of Chk is induced in primary breast cancer cells upon stimulation with heregulin but not in normal breast tissues. The binding of Chk to ErbB-2/neu receptor-type tyrosine kinase induces inhibition of Src kinase activity and of cancer cell growth [14], [18], suggesting that Chk plays a role in signal transduction in other cell types besides hematopoietic and neuronal cells. The distinct intracellular localization of Chk may be critical for its function. We wished, therefore, to explore the effect of nuclear localization of Chk on the functions of the nucleus.