The Study On The Function Of Lck Kinase In L-selectin Signaling

Leukocyte recruitment from the bloodstream to a site of inflammation entails a cascade of cellular adhesive events, including tethering, rolling, adhesion and transmigration. This process is controlled strictly by three families of adhesive molecules expressed on leukocytes and endothelial cells, and selectin family is responsible for the initial tethering and rolling of leukocytes. As a member of selectin family, L-selectin constitutively expresses on the top of microvilli and appears to be the first receptor for the early kinetics of leukocyte-endothelial cell interaction. Besides, L-selectin contributes in leukocyte adhesion through its signaling properties. L-selectin engagement with antibody cross-linking could trigger actin cytoskeleton rearrangement, cell shape change, superoxide generation, increased gene expression of cytokine TNF-αand IL-8, and so on. We still do not know how L-selectin orchestrates those cytoplasmic proteins to transduce signals. Lck kinase is a member of the Src family of non-receptor protein tyrosine kinases, which is expressed primarily in T lymphocytes and plays an important role in T cell activation and development. Cross-linking L-selectin with antibody induces the activation of Lck kinase, so we suppose that Lck kinase not only is involved in L-selectin signaling, but also plays a critical role in the process. In this paper, we focus on the Lck kinase function in regulating L-selectin signaling induced by sulfatides engagement.In Jurkat T cells, we find that L-selectin ligation with sulfatides induces rapid and robust tyrosine phosphorylation of Lck kinase, while PP2 (Lck specific inhibitor) significantly inhibits actin cytoskeleton rearrangement induced by L-selectin engagement and L-selectin-mediated Jurkat T cell rolling on HUVECs (human umbilical vein endothelial cells). These results suggest Lck kinase is involved in L-selectin signaling and plays an important role. Through the direct interaction with L-selectin cytoplasmic tail, activated Lck kinase could phosphorylate cytoplasmic tail in a minute promoting signal transduction. The only tyrosine, Tyr372, of cytoplasmic tail of L-selectin has an effect on the direct interaction with Lck kinase, for its mutation dramatically inhibits this association. After sulfatides stimulation for 2 min, the interaction between L-selectin and Lck kinase attenuates, suggesting that Lck kinase detaches from L-selectin cytoplasmic tail. For the dynamics of interaction, L-selectin could bind different cytosolic signaling proteins using its cytoplasmic tail composed of 17 amino acids.Through the direct interaction with c-Abl and ZAP-70 kinases via its SH3 and SH2 domains, Lck kinase organizes a signaling complex at the cytoplasmic tail of L-selectin. After sulfatides engagement, Lck kinase activated prior to c-Abl kinase. PP2 (Lck specific inhibitor) could dramatically inhibit the phosphorylation and kinase activity of c-Abl kinase, which also nearly completely decreased the phosphorylation of total and Tyr319 of ZAP-70 kinase. STI571 (c-Abl specific inhibitor) and Piceatannol (ZAP-70 specific inhibitor) play no role on the phosphorylation of Lck kinase. These results indicate that Lck kinase is the first kinase to be activated and regulate the kinase activity of both c-Abl and ZAP-70 kinases in L-selectin signaling pathway.In the cells with Lck knockdown by siRNA treatment, L-selectin signaling is severely suppressed, as indicated by reduced phosphorylation of L-selectin, c-Abl kinase and ZAP-70 kinase and by decreased number of Jurkat T cell rolling on HUVECs. Re-expression of wild-type or constitutively active but not kinase-dead murine Lck completely rescue the phosphorylation, suggesting the kinase activity of Lck is important for L-selectin signaling. Re-expression of SH2 domain mutant has no effect on protein phosphorylation but partly rescues the cell rolling, while re-expression of SH3 domain mutant has the opposite role, indicating that SH2 and SH3 domains of Lck kinase perform different signaling functions.In summary, Lck kinase plays a critical role in L-selectin signaling induced by sulfatides engagement. Through the direct interaction with L-selectin cytoplasmic tail, Lck kinase accepts and amplifies L-selectin signal. On the other hand, Lck kinase transduces signals quickly and efficiently by binding c-Abl and ZAP-70 kinases via its SH3 and SH2 domains. Meanwhile, because of the dynamic interaction with Lck kinase, L-selectin could bind other cytosolic proteins to transducer signals in other signaling pathway.