The Apoptotic Mechanism of Death-associated Protein Kinase and its Regulation by Protein Tyrosine Phosphatase LAR

• Main Authors: Won-Jing Wang, 王琬菁
• Other Authors: Ruey-Hwa Chen
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/07819540476327236497

博士 === 國立臺灣大學 === 分子醫學研究所 === 93 === Death associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptotic systems. In this thesis, we studied the apoptotic mechanism of DAPK and its signaling and functional crosstalk with a phosphotyrosine phosphatase. In the first part of study, we demonstrated that DAPK suppresses integrin-mediated cell adhesion by down-regulating integrin activity through an inside-out mechanism. This adhesion-inhibitory effect of DAPK blocks integrin survival signals and up-regulates p53 protein, thereby inducing apoptosis. In support of this notion, we demonstrated that enforced activation of integrin survival pathways from either integrin itself or its downstream effector FAK abolishes the apoptotic effect of DAPK, and that DAPK can no longer induce apoptosis in the anoikis resistant cells. Thus, our study unravels that apoptotic mechanism of DAPK and identifies DAPK as an inducer of anoikis. In the second part of this thesis, we searched for DAPK interaction partners as part of our work to dissect DAPK signaling network. A yeast two-hybrid screen using the anykrin-repeat domain of DAPK as bait identified the leukocyte common antigen related tyrosine phosphatase (LAR) as a DAPK interacting protein. We showed that DAPK interacts specifically with LAR, through the ankyrin repeats 3-6 of DAPK and the phosphatase domain of LAR. The higher binding affinity of DAPK towards a substrate-trapping mutant of LAR suggests that DAPK functions as a substrate of LAR. Indeed, DAPK can be efficiently tyrosine-dephosphorylated by LAR both in vitro and in vivo, and this dephopshorylation event occurs at Y491/492 residues of DAPK. Furthermore, we demonstrated that LAR up-regulates the catalytic activity of DAPK through an Y491/492-dependent manner. As a consequence, the various biological activities of DAPK, such as anti-adhesion and apoptosis induction, are significantly promoted by overexpression of LAR. In contrast, knockdown of endogenous LAR by RNA interference technique results in an elevation of DAPK tyrosine phosphorylation at Y491/492 and inhibition of DAPK biological functions. These data indicate that LAR functions as a novel activator of DAPK through dephosphorylating DAPK at Y491/492. The uncovering of DAPK-LAR interplay and DAPK apoptotic mechanism in this thesis would shed light on the molecular mechanisms of DAPK signaling and biological functions.