Structural Basis of Phosphorylation and Alternative Splicing in Dynein Regulation

Oregon State University, Corvallis OR

Investigators

Abstract

TITLE: Structural Basis of Phosphorylation and Alternative Splicing in Dynein Regulation Living systems use biological motors to transport materials into cells in specific directions along train-like tracks called microtubules. One such motor is dynein which is essential for many cellular processes including transport of chromosomes during cell division, movement of vesicles containing nutrients, and in neuronal migration. How dynein knows when and what cargo to transport requires interactions with other proteins that control dynein function. These interactions are regulated by modification in the protein sequence in regions of dynein that lack regular unique three-dimensional structure and are referred to as intrinsically disordered proteins. Expected results will specifically show how these modifications help in selecting the binding partners, and subsequently what cargo to transport. Understanding this system will ultimately be the basis for the design of synthetic nano-machines that can transport materials on demand. This work will facilitate the training of undergraduate and graduate students in the practice of cutting-edge research. In addition, the PI and her team will impact progress in other disciplines at Oregon State University and the region by providing access to biophysical instrumentation, by initiating and organizing an annual NMR workshop and an annual regional one-day conference focused on biophysics, and by implementing hands on demonstrations and science activities of varying complexity to K-12 area schools that include models/animations of protein disorder and structural changes for large-scale demonstrations at the Oregon Museum of Science. This research addresses how phosphorylation and intrinsic disorder in one subunit of dynein modulate the interaction of dynein with multiple regulatory proteins including dynactin, whose interaction occurs in all eukaryotes and plays an especially important role in neurons, and NudE, a nuclear distribution protein which is essential in diverse processes including kinetochore and centrosome migration. The intrinsically disordered N-terminal 300-amino acid domain of dynein intermediate chain (IC) is responsible for these interactions and is also the hub of most dynein activity. While there is ample evidence for phosphorylation and isoform expression as key to dynein regulation, there are no studies of the molecular mechanisms underlying their effects. This project will specifically focus on: 1) How IC phosphorylation affects the interaction of tissue-specific isoforms of IC with dynactin, 2) How IC phosphorylation selects between regulatory proteins dynactin and NudE, and 3) How IC regulation differs among mammals, Drosophila, and yeast. These outstanding questions about dynein regulation will be addressed using a combination of state-of-the-art NMR, isothermal titration calorimetry, synthetic biology, computational methods, and in vivo-based assays and will elucidate how phosphorylation and alternative splicing of intrinsically disordered proteins in general govern their molecular function and ultimately affect the behavior of entire protein interaction networks. Significantly, the use of proteins from yeast, Drosophila and mammalian origin, assesses the different IC processes that evolved for inducing structural changes within highly disordered regions, underscoring the strategic role for protein disorder and IC isoforms in regulation of dynein function.