ABSTRACT
Zeta chain-associated protein kinase 70 (ZAP-70) is a non-receptor tyrosine kinase that interacts with the activated T-cell receptor to transduce downstream signals, and thus plays an important role in the adaptive immune system. The biphosphorylated immunotyrosine-based activation motifs (ITAM-Y2P) binds to the N-SH2 and C-SH2 domains of ZAP-70 to promote the activation of ZAP-70. The present study explores molecular mechanisms of allosteric inactivation of ZAP-70 induced by the hot spot W165C mutation through atomically detailed molecular dynamics simulation approaches. We report microsecond-length simulations of two states of the tandem SH2 domains of ZAP-70 in complex with the ITAM-Y2P motif, including the wild-type and W165C mutant. Extensive analysis of local flexibility and dynamical correlated motions show that W165C mutation changes coupled motions of protein domains and community networks. The binding affinities of the ITAM-Y2P motif to the wild-type and W165C mutant of ZAP-70 are predicted using binding free energy calculations. The results suggest that the driving force to decrease the binding affinity in the W165C mutant derives from the difference in the protein-protein electrostatic interactions. Moreover, the per-residue free energy decomposition unravels that the contributions from residues in the phosphorylated Tyr315 (pY315) binding site, in particular pY315 of ITAM-Y2P, and Arg43, Tyr240 of ZAP-70, are the key determinants for the loss of binding affinity. This study may insights into our understanding of the pathological mechanism of ZAP-70.Communicated by Ramaswamy H. Sarma.
ABSTRACT
Gallbladder carcinoma (GBC) represents the most common fatal tumors of the biliary tract. The 3-year or 5-year survival rate for patients with this disease are 30 and 5%, respectively. Liver kinase B1 (LKB1), a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK) necessary for maintaining cell metabolism and energy homeostasis, has been found to be an important tumor suppressor gene in recent years, and its inactivation has also found to be closely associated with tumor growth, metastasis and cancer stem cell (CSC) proliferation. Nevertheless, the function of LKB1 in GBC remains unclear. In this study, we found that the expression of LKB1 in GBC tissues was decreased compared with that in non-cancerous tissues. LKB1 overexpression suppressed the proliferation, metastasis and expansion of GBC CSCs. Mechanically, LKB1 suppressed GBC cell progression via the JAK/signal transducer and activator of transcription 3 (STAT3) pathway. The use of the JAK2 inhibitor, AZD1480, attenuated the suppressive effects of LKB1 overexpression on the growth, metastasis and self-renewal ability of the GBC cells, which further demonstrated that JAK/STAT3 was involved in the LKB1-induced suppression of GBC cell growth, metastasis and self-renewal ability. More importantly, the decreased expression of LKB1 was a predictor of a poor prognosis of patients with GBC. On the whole, our data indicate that LKB1 inhibits GBC cell growth, metastasis and self-renewal ability by disrupting JAK/STAT3 signaling, and may thus prove to be a novel prognostic biomarker for patients with GBC.
