Date of Award

12-15-2014

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Deanna Smith

Abstract

Dynein is a microtubule minus-end directed molecular motor, participating in a broad range of cellular functions, such as organelle transport, cell migration and mitosis. Dynein dysfunction is linked to many diseases including ALS, schizophrenia, Alzheimer’s disease, Parkinson’s disease and cancer. The mechanism of dynein regulation is largely unknown. We have provided evidence that glycogen synthase kinase 3β (GSK-3β) directly regulates dynein in both neurons and non-neuronal cells. GSK-3β interacts with and phosphorylates dynein in vitro. Dynein phosphorylation by GSK-3β reduces its interaction with Ndel1, a regulator contributing to dynein force generation. Dynein motility is stimulated both by pharmacological GSK-3β inhibitors and by enhanced insulin signaling that leads to GSK-3β inactivation. Thus our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition. There is considerable debate over whether thiazolidinediones, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, are chemopreventive or carcinogenic during the development of colorectal cancer, where mutations in adenomatous polyposis coli (APC) often occur. We have demonstrated that the interplay of APC and dynein may be important for PPAR-γ signaling to regulate cancer development. Dynein and APC physically interact with each other, which is positively regulated by GSK-3β. Rosiglitazone increases dynein activity and cell migration in wild type (WT) but not in Apc (min/+) cells, and causes spindle misorientation in Apc (min/+) but not in WT cells. We provide evidence that this involves different PI3K/AKT/GSK-3β signaling responses to rosiglitazone between WT and Apc (min/+) cells and the disruption of the dynein-APC interaction by ApcMin mutation. Dynein intermediate chain (IC) is essential for dynein assembly and mediates the interactions of dynein to regulators. To dissect how GSK-3β and APC regulate dynein, we developed a mass spectrometry (MS)-based systematic method to map phosphorylation sites on IC. We identified that T154, S88 and T89 on IC-2C are targeted by GSK-3β using MS and mutagenesis. S88 and T89 are conserved in all mouse IC isoforms and ICs from all other mammalian species. Furthermore, we demonstrate that S87 or T88 on IC-1B (corresponding to S88 or T89 on IC-2C) from both mouse and rat are targeted by GSK-3β using MS. The method has the potential to be applied to identify other bona fide substrates of GSK-3β or other kinases.

Rights

© 2014, Feng Gao

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