FLA8/KIF3B Phosphorylation Regulates Kinesin-II Interaction with IFT-B to Control IFT Entry and Turnaround

Abstract

The assembly and maintenance of cilia depends on intraflagellar transport (IFT). Activated IFT motor kinesin-II enters the cilium with loaded IFT particles comprising IFT-A and IFT-B complexes. At the ciliary tip, kinesin-II becomes inactivated, and IFT particles are released. Moreover, the rate of IFT entry is dynamically regulated during cilium assembly. However, the regulatory mechanism of IFT entry and loading/unloading of IFT particles remains elusive. We show that the kinesin-II motor subunit FLA8, a homolog of KIF3B, is phosphorylated on the conserved S663 by a calcium-dependent kinase in Chlamydomonas. This phosphorylation disrupts the interaction between kinesin-II and IFT-B, inactivates kinesin-II and inhibits IFT entry, and is also required for IFT-B unloading at the ciliary tip. Furthermore, our data suggest that the IFT entry rate is controlled by regulation of the cellular level of phosphorylated FLA8. Therefore, FLA8 phosphorylation acts as a molecular switch to control IFT entry and turnaround.

Gates for soluble and membrane proteins, and two trafficking systems (IFT and LIFT), establish a dynamic ciliary signaling compartment

“…The distal segment may contain different proteins and often, a different microtubule structure (singlets). Lastly, the ciliary tip includes factors for IFT turnaround and microtubule dynamics [7,8].…”

Functional exploration of the IFT-A complex in intraflagellar transport and ciliogenesis

“…They are returned from the ciliary tip to the base via cytoplasmic dynein 1b/2 (retrograde IFT) [6–9]. There are thus various points in the process of IFT where it might be regulated: IFT entry and exit of cilia, turnaround at the ciliary tip, anterograde and retrograde transport of IFT[10–13]. …”

The Intraflagellar Transport Machinery

“…After reaching the tip, kinesin inactivation and dynein activation are a prerequisite for retrograde IFT. Interaction studies between IFT complex proteins and motors have so far not been very conclusive and, in some cases, contradictory (Baker et al 2003;Qin et al 2004;Follit et al 2009;Liang et al 2014). A direct interaction between heterotrimeric kinesin II and IFT20 was proposed for the mouse proteins based on a yeast two-hybrid assay (Baker et al 2003), but this could not be confirmed in an independent study (Follit et al 2009), and might have been a false positive interaction caused by coexpression of two coiled-coil proteins in the absence of their native binding partners.…”