The roles of GSK-3beta and APC in cytoplasmic dynein regulation

Dynein is a microtubule-based retrograde molecular motor, participating in a broad range of cellular functions, such as organelle transport, cell migration and mitosis. Dynein dysfunction causes 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 3beta (GSK-3beta) directly regulates dynein in both neurons and non-neuronal cells. GSK-3beta interacts and phosphorylates dynein in vitro. Dynein phosphorylation by GSK-3beta reduces its interaction with Ndel1, a regulator contributing to dynein force generation. Dynein motility is stimulated both by pharmacological GSK-3beta inhibitors and by enhanced insulin signaling that leads to GSK-3beta 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-gamma) 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-gamma signaling to regulate cancer development. Dynein and APC physically interact with each other, which is positively regulated by GSK-3beta. Rosiglitazone increases dynein activity and cell migration in wide type (WT) but not in Apc (min/+) cells, and causes spindle misorientation in Apc (min/+) but not in WT cells. We provides evidence that this may be caused by different PI3K/AKT/GSK-3beta 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-3beta and APC regulates dynein, we develop a mass spectrometry (MS)-based systematic method to map phosphorylation sites on IC. We identify T154, S88 or T89 on IC-2C are targeted by GSK-3beta using MS and mutagenesis. S88 and T89 are conserved in all mouse IC isoforms and ICs from 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 is targeted by GSK-3beta using MS. The method has the potential to be applied to identify other bona fide substrates of GSK-3beta or other kinases.