Kinesins and dyneins are microtubule-based motor proteins that drive diverse cellular processes, including mitosis, intracellular transport of vesicles, and ciliary function. The importance of motor protein function is reflected by the plethora of human diseases associated with motor malfunction, ranging from developmental defects to cancer, neurodegenerative diseases, and obesity. To ensure physiological function, the activity of motor proteins has to be precisely controlled. One such regulation mechanism that controls kinesins is autoinhibition. Many kinesins possess a hinge in the stalk domain, allowing the kinesin to fold back onto itself so that the tail domains can bind to and thereby inhibit the motor domains. This autoinhibitory interaction also prevents cargo binding and motor association with microtubules. It has been proposed that autoinhibition is a general mechanism by which most kinesin motors are regulated, but the molecular mechanism of autoinhibition has only been elucidated for several kinesins. Since kinesin tail domains are tailored to the transport of specific cargo, they are structurally divergent between different kinesin types. Thus, the interactions that enable autoinhibition are distinct between different members of the kinesin family. In this proposal, we will study the regulation of the heterotrimeric kinesin-2 (KIF3A/KIF3B and associated subunit KAP). KIF3A/KIF3B drives several cellular processes, including the transport of N- cadherin for cell adhesion and vesicle transport in the cytoplasm and axon of neurons. It also drives a specialized transport process called intraflagellar transport (IFT), which is necessary for the creation (ciliogenesis) and maintenance of cilia. Cilia are microtubule-based, hair-like, plasma membrane protrusions, with important sensing functions. They are found on the surface of most if not all human cells and are essential for mammalian development and adult physiology. In previous work, the PI generated chimeric KIF3A/KIF3B constructs and found that these motors strongly accumulated in the cell periphery, indicative of lost autoinhibition. The autoinhibition mechanism of the KIF3A/KIF3B motor is not known, but this finding suggests that specific interactions between the stalk-tail and motor domains mediate autoinhibition in this motor. The chimeric KIF3A/KIF3B motors were also not able to drive ciliogenesis in Kif3a-/-;Kif3b-/- double knockout cells. This indicates that intact autoinhibition is necessary for KIF3A/KIF3B to drive critical physiological processes such as IFT. In this proposal, we will use a combination of biochemical & cellular assays, protein & genome engineering, and fluorescence microscopy, to delineate the interactions that mediate autoinhibition in the KIF3A/KIF3B motor. We will also study the effect of phosphorylation on motor activity and cargo binding. The work laid out in this project will shed light on the function and regulation of kinesin motors and thereby promote the development of therapies aimed at alleviating or curing motor protein-associated human diseases.
(lay abstract) Cancer, developmental defects, and neurodegenerative diseases all can be caused by a malfunction of cellular nanomachines, called motor proteins. This proposal will determine how the activity of these motor proteins is regulated in order for them to perform their cellular functions. This work will contribute to our understanding of motor protein biology, thus leading to novel directions for therapeutic approaches to alleviate human diseases.
