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Activation of intracellular transport by relieving KIF1C autoinhibition

Nida Siddiqui, Alice Bachmann, Alexander J Zwetsloot, Hamdi Hussain, Daniel Roth, Irina Kaverina, Anne Straube

Preprint posted on December 06, 2018 https://www.biorxiv.org/content/early/2018/12/06/488049

Straightening out: KIF1C is activated by the binding of scaffolding proteins PTPN21 or HOOK3

Selected by Ben Craske, Thibault Legal and Toni McHugh

Categories: biochemistry, cell biology

Context:

The molecular motor KIF1C is a fast plus end-directed kinesin which is important for cargo transport in several cell types, delivering dense core vesicles to the dendrites of neurons and trafficking integrins to the actin-rich adhesions of macrophages and smooth muscle cells. It has been suggested that like other kinesin-3 motors KIF1C may cooperate with dynein to enable bidirectional cargo transport, but how this mechanism is regulated is unclear. Most kinesin-3 motors are inactive, diffusive monomers which dimerise upon cargo binding, activating their processive movement. This mechanism of autoregulation is distinct to kinesin-1 and kinesin-7 motors, which are regulated through direct interactions between the tail domains and the motor and neck regions, inactivating them until cargo binding relieves inhibition.

Key Findings:

In this paper Siddiqui et al. show that unlike other Kinesin-3 motors, purified KIF1C is intrinsically dimeric. In high salt concentrations, KIF1C adopts a more elongated conformation than in physiological conditions which suggests that the non-motor regions may fold into a compact, self-interacting structure. Using crosslinking mass spectrometry the authors show that the microtubule-binding interface of the motor domain interacts with the FHA domain and the third coiled-coil in the stalk. This indicates that such self-interactions may block the motor domain from associating with the microtubule and subsequent processivity(Fig. a). They also find that depletion of a previously characterized KIF1C interactor, PTPN21 (protein tyrosine phosphatase N21), phenocopies KIF1C depletion resulting in a reduction in the frequency of podosome formation. Interestingly, rescue is not dependent on the catalytic activity of PTPN21, as a minimal region of PTPN21 containing only the FERM domain (Fig. a) was sufficient to rescue the phenotype, suggesting that the scaffolding function was important for KIF1C activation. By imaging vesicle-transport in both RPE1 cells and primary hippocampal neurons Siddiqui et al. show that the reduction of both plus and minus end directional transport observed upon partial depletion of KIF1C can be rescued by over expression of the PTPN21 FERM domain. This demonstrates that this activation mechanism is common between different cell types.

 

 

Using in vitro reconstitutions, the authors show that the PTPN21 FERM domain is able to activate KIF1C. This is shown by an increase in both landing rate and frequency of motor movement. Using crosslinking mass spectrometry, they find that PTPN21 FERM domain forms crosslinks with the FHA/coiled-coil 3 (CC3) region of KIF1C responsible for its auto inhibition. To further confirm that this region is important for autoregulation, they generated a KIF1C_delCC3 mutant lacking the CC3 domain. Single molecule motility assays indicate that KIF1C_delCC3 has a higher landing rate than KIF1C in vitro. Additionally, the landing rate of the mutant is no longer increased by inclusion of PTPN21-FERM, indicating that inhibition is fully relieved in the absence of the coiled-coil 3 domain.

Finally, Siddiqui et al. use a BioID to look for other KIF1C stalk interactors. The authors identified 240 proteins which are pulled down with KIF1C-BioID2 but not KIF1C_del623-825-BioID2. Interestingly, their top hit is Hook3, a dynein/dynactin activator that may be involved in mediating bidirectional cargo transport. In vitro they demonstrate that Hook3 is able to activate KIF1C in a similar fashion to PTPN21 and show co-transport of Hook3-Alexa647 with KIF1C in their single molecule kymographs.

Overall this paper shows that KIF1C is a dimeric kinesin-3 motor and is autoinhibited through a direct interaction between the FHA/CC3 stalk region and the microtubule-binding region in the motor domain.  This inactive, compact structure can be relieved through interaction with binding partners such as PTPN21 or Hook3, increasing the landing rate of the motor on microtubules in vitro and enabling bidirectional cargo transport in cells (Fig. a,b).

Open questions:

Were there any other interesting candidates in the BioID screen? For example do any other dynein activators appear in the screen?

If Hook3 can simultaneously bind and activate both KIF1C and dynein through non-overlapping binding sites, how might this work to regulate switching from plus to minus end directed movement?

Why we chose this paper:

We chose this preprint as we are interested in the different mechanisms underpinning kinesin regulation and how cargo binding may mediate the activation of such motors through structural changes. This paper demonstrates that KIF1C is regulated in a distinct manner compared to other kinesin-3 family members and uses a combination of biochemistry, in vitro reconstitutions and cell biology to illustrate how inhibition is relieved by binding partners.

 

Tags: hook3, kif1c, kinesin, ptpn21, transport

Posted on: 7th January 2019

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  • Author's response

    Dr Anne Straube shared

    Were there any other interesting candidates in the BioID screen? For example do any other dynein activators appear in the screen?

    We do find the KIF1C cargo beta1-integrin (Theisen et al. (2012) Directional persistence of migrating cells requires Kif1C-mediated stabilization of trailing adhesions. Dev Cell 23:1153-1166) in the full length BioID, but not in the deletion, which is consistent with the idea that Hook3 might mediate integrin transport by KIF1C (and dynein). However, other dynein adapters such as BicD2 were found to similar levels with full length and the deletion, which is consistent with previous findings that BicD-related 1 binds to the C-terminal Proline-rich domain of KIF1C (Schlager et al (2010), Pericentrosomal targeting of Rab6 secretory vesicles by Bicaudal-D-related protein 1 (BICDR-1) regulates neuritogenesis. EMBO J 29:1637-1651). Consequently, we also find dynein heavy chain with both BioID constructs.

    If Hook3 can simultaneously bind and activate both KIF1C and dynein through non-overlapping binding sites, how might this work to regulate switching from plus to minus end directed movement?

    This is an exciting question we and others are currently working on. Agnieszka Kendrick from Samara Reck-Peterson’s lab presented her findings that Hook3 simultaneously binds to dynein/dynactin and KIF1C and that these complexes move processively to either end of the microtubule, but not undergo directional switching in vitro at the recent ASCB-EMBO meeting (Kendrick et al. (2018), HOOK3 is a Scaffold for Opposite Polarity Motors. Mol Biol Cell 29: 3063, #M75). Thus the current idea is that directional switching is regulated in cells and might require additional factors that regulate the activity of each motor.

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