Biochemical, structural and functional characterization of the light chains of the microtubule-based motor dynein.

摘要

Cytoplasmic dynein is a large multisubunit motor protein that moves various cargoes toward the minus end of microtubules. It is composed of a number of different subunits including three light chain families: 8 kDa dynein light chain (DLC8), 14 kDa dynein light chain (Tctex1 and rp3) and roadblock light chain. The incorporation of the DLC8, Tctex1 and rp3 light chains into the cytoplasmic dynein complex has been determined by in vitro "pull-down" assays. We further showed that both DLC8 and Tctex1 interact specially with a number of functionally unrelated targets, indicating that they are likely to be multifaceted regulatory proteins. Two consensus sequences were identified in DLC8-interacting proteins. One of the consensus binding motif has amino acid sequence of "K/RXTQT" which is present in dynein intermediate chain (DIC), proapoptotic Bim, several viral proteins like rabies P protein and BS69. The other sequence is "GIQVD" which is found in GKAP, nNOS, and 53BP1. I also found that various Tctex-1 target proteins contain a consensus motif of "R/KR/KXXR/K". The solution structure of Tctex1 was determined using multidimensional NMR spectroscopy. Even though DLC8 and Tctex1 share no amino acid sequence homology, our results suggested that Tctex1 adopts a structure similar to DLC8.;Two novel DLC8-binding proteins paxillin and 53BP1 were identified by yeast two-hybrid screening. The functional significance of the interaction between DLC8 and these two newly identified DLC8-binding targets were subsequently studied. The DLC8/paxillin interaction may play important roles in cell spreading and the DLC8/53BP1 interaction may involve in p53 nuclear accumulation in response to DNA damage.