Biochemical and Biophysical Research Communications
Tyro3 carboxyl terminal region confers stability and contains the autophosphorylation sites
Introduction
Tyro3, Axl, and MerTK comprise a receptor tyrosine kinase family (TAM) which has been implicated in cell proliferation, survival, migration and tumor angiogenesis upon stimulation by extracellular phospholipid ligands such as GAS6 [1]; [2]; [3]; [4]; [5]; [6]. Abnormal expression of Axl and MerTK has been widely reported in many cancer cells [7]; [8]; [9]; [10]; [11]. Compared to Axl and MerTK, Tyro3 is the least studied although recently accumulating evidence revealed that the expression of Tyro3 is elevated in several cancer cells such as ovarian cancer, hepatocellular carcinoma, colon cancer and melanoma [12]; [13]; [14]; [15]; [16]. For example, Lee reported that overexpression of Tyro3 leads to a resistance in ovarian cancer cells to chemotherapeutic drug taxol [12]. Thus Tyro3 is being considered as a potential therapeutic target in patients with cancer. To this point, we have found that Tyro3 can promote invasiveness of melanoma cells {Shao, 2017, submitted}. How these kinases actuate signal transduction downstream from the membrane is still ill-defined.
The three TAM family members share three types of conserved domains including two extracellular fibronectin type III (FNIII), two immunoglobulin-like domains and a unique kinase domain. The TAM family contains a conserved sequence, KW (I/L)A(I/L)ES which does not exist within the kinase domain of other RTKs [17]. Ligand binding to RTK receptors induces receptor dimerization and subsequent trans-autophosphorylation of tyrosine residues within the intracellular domain of RTK receptors which results in an increase of catalytic efficiency so that other substrates can be phosphorylated and a recruitment of signaling molecules containing SH2, PTB, or other phosphotyrosine-binding domains to docking sites of tyrosine-phosphorylated RTKs and other proteins. Human MerTK autophosphorylates at Y749, Y753 and Y754 [18]. By analogy, the tyrosines at Y779, Y821 and Y866 have been proposed as potential autophosphorylation sites of human Axl although there were no clear evidence to support that these three tyrosine residues are indeed autophosphorylation sites [19]. The autophosphorylation sites of Tyro3 have been suggested to localize within its C-terminal region [20]. In the present study, we identified Y723 and Y756 as two novel autophosphorylation sites of murine Tyro3. Importantly, we also found that Tyro3 stability is conferred by the domain beyond the autophosphorylation sites, suggesting a mechanism by which tumor-associated signaling could be regulated.
Section snippets
Cell culture and reagents
Fibroblasts NR6WT were cultured in alpha-MEM media supplemented with 1x sodium pyruvate, 1x non-essential amino acids, 1x pen/strep antibiotics, 1x l-glutamine, and 7.5% fetal bovine serum. Melanoma cell line WM1158 was cultured in DMEM (1 gL−1 glucose): L15 3:1 medium supplemented with 10% fetal bovine serum and 1x pen/strip antibiotics. Transfection reagent xFect was purchased from Clonetech Life Technologies (Grand Island, NY). Monoclonal phospho-tyrosine antibody (p-Tyr-100) was purchased
Mapping of Tyro3 kinase domain
The autophosphorylation of TAM receptors has been previously reported [18]; [19]. Braunger et al. revealed that replacement of lysine with an arginine in the ATP binding pocket of human Axl (K567 > R567) quenched its autophosphorylation in 293 cells [19]. By aligning the amino acids sequence of human Axl and murine Tyro3, we found that human Axl K567 maps to murine Tyro3 K540. However, previous reports have K536 being designated the ATP binding site of murine Tyro3 in Rat2 cells [22].
Discussion
Herein, we mapped the phosphorylation functional sites of the receptor protein tyrosine kinase Tyro3. We found that the ATP acceptor is the second one in the nucleoside binding pocket, and that autophosphorylation occurs on two tyrosines in the unique carboxyl terminal region after the kinase domain, at Y723 and Y756. It should be noted that we did not demonstrate intra-molecular autophosphorylation per se, but rather tyrosyl-phosphorylation that occurs upon forced overexpression. That this was
Acknowledgements
This work was supported, in whole or in part, by grants from the National Institutes of Health (NIGMS) (GM69668 and GM63569). The Pittsburgh VA provided in kind support.
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Tyro3-mediated phosphorylation of ACTN4 at tyrosines is FAK-dependent and decreases susceptibility to cleavage by m-Calpain
2018, International Journal of Biochemistry and Cell BiologyCitation Excerpt :We found that active Gas6 did not increase the observed Tyro3-mediated phosphorylation of ACTN4 in NR6WT cells (data not shown). This is most probably due to the saturated autophosphorylation of Tyro3 (Shao et al., 2017) that cannot be enhanced by the binding of Gas6 to Tyro3; this was determined by failure of Tyro3 clustering to increase the phosphorylation level (data not shown). However, the activation of Tyro3 is required for the phosphorylation of ACTN4 at tyrosine 11 and 13 because K540R, a kinase dead mutant of Tyro3 was no longer able to trigger ACTN4 phosphorylation (Shao et al., 2017).