The regulation of KIF3A motor subunit by phosphorylation

• Main Author: Chen, Kunning
• Other Authors: Koh Cheng Gee
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/73335
• Institution: Nanyang Technological University

The Kinesin-2 family member KIF3 forms a heterotrimeric complex comprising the motor subunits KIF3A, KIF3B and the cargo carrying subunit KAP3. N-cadherin and b-catenin are reported to be cargoes of KIF3 motor. Serine-689 residue at the C-terminal of KIF3A has been identified as a major phosphorylation site significant for the transport of cargoes. In an attempt to find out the effect of phosphorylation on KIF3A’s function, serine-689 was mutated to alanine (S689A) to preclude phosphorylation, and to aspartic acid (S689D) to mimic continuous phosphorylation. In this study, we showed that overexpressing S689A mutant impaired the transportation of N-cadherin and b-catenin to the cell-cell junction, indicating that the phosphorylation status of serine-689 residue plays a crucial role in the intracellular KIF3-mediated cargo transport. We also identified that the interaction between the KIF3A motor and tail domain was relieved by Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in vitro and in vivo by different assays. Our results also suggest that KIF3A’s ATPase activity was inhibited by the motor-tail interaction. Phosphorylation on serine-689 residue relieves the motor-tail interaction to allow recovery of the motor’s ATPase hydrolysis activity. Computational simulation of KIF3A motor-tail complex under different phosphorylation states also supports our observations. Based on these findings, we hypothesize that KIF3A adopts a folded conformation resulting in an autoinhibited state, and phosphorylation on the serine-689 residue at the C-terminal tail domain of KIF3A subunit will relieve this autoinhibition and induce a conformational change to expose the microtubule and ATP binding regions at the motor domain. The activated KIF3 motor can thus slide on microtubules to transport its cargoes. Detailed structural information is needed to explain KIF3A’s mechanisms. In this study, a low-resolution structure of KIF3A’s motor domain was reconstructed by Small-angle X-ray scattering (SAXS) data. The screening of the proper protein crystal growing conditions for X-ray crystallography is ongoing. Promising conditions for crystallization will be discussed.