Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin α1

Abstract

Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin α1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/ KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PIð3,4ÞP 2 ). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.kinesin | forkhead-associated domain | vesicle transport | phosphatidylinositol triphosphate | neuronal development N eurons are highly polarized cells that typically feature one long axon and several shorter dendrites. Newborn neurons initially develop immature neurites that undergo constant, random growth and retraction regulated by positive and negative feedback signals (1), and become polarized when this balance is shifted so that one neurite becomes an axon and the others become dendrites. Accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), which is synthesized by phosphoinositide 3-kinase (PI3K) and catabolized by the phosphatase PTEN (2) at the distal end of one neurite, induces axonal development (2, 3). However, an alternative pathway for PIP3 accumulation is the transport of PIP3-containing vesicles by KIF13B and the PIP3-binding protein centaurin α1 (CENTA1) (4).KIF13B is a kinesin-3 subfamily member containing an N-terminal motor domain with ATPase activity that drives movement along the microtubule and a forkhead-associated (FHA) domain, the only known phosphothreonine (pThr)-specific recognition domain (5-8). KIF13B also contains a long coiled-coil domain and a C-terminal glycine-rich microtubule-binding domain (9), both of which function in dimer formation (10). KIF13B does not interact with PIP3-containing vesicles directly; rather, it binds via its FHA domain to CENTA1 (4), which contains an ArfGAP domain that inactivates Arf6 (11) and two pleckstrin homology (PH) domains that interact with PIP3 (12-15). CENTA1 is associated with presynaptic vesicle structures (16), and its expression is elevated in the neurons of Alzheimer's patients (17).To understand how CENTA1 and PIP lipids interact, we solved the structures of lipid-free and lipid-bound forms of CENTA1 and confirmed binding specificity using a PIP array assay. We found that the first PH domain of CENTA1 binds PIP3, while the second binds phosphatidylinositol (3,4)-bisphophate (PIð3;4ÞP 2 ) and PIP3. We also solved the structure of the KIF1...

Cyclin-Dependent Kinase 5 Controls TRPV1 Membrane Trafficking and the Heat Sensitivity of Nociceptors through KIF13B

“…In another study, KIF13B was found to transport phosphatidylinositol-3,4,5-triphosphate (PIP 3 )-containing vesicles to neurite endings via the adaptor protein centaurin ␣1/PIP 3 binding protein (Horiguchi et al, 2006;Tong et al, 2010), and the FHA domain is just the domain that directly interacts with centaurin ␣1, which, in turn, specifically binds to PIP 3 . In the case of TRPV1, the motor associates with the cargo in a phosphorylation-dependent manner; however, it is not known whether the FHA Figure 8.…”

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

“…4 Several FHA domains have been shown to mediate bipartite interactions. For instance, the FHA domain of Dun1 requires the simultaneous interaction with two pThr residues to recognize its target (21), and KIF13B interacts simultaneously with the ArfGAP and PH1 domains of CENTA1 (38). Therefore, FHA domains have been compared with logic gates able to integrate multiple inputs and generate a single output (22).…”

Extending the Interaction Repertoire of FHA and BRCT Domains

“…In this case, the H-BRCT domain of Dbf4 could interact with the FHA1 domain of Rad53 in a phos-phorylation-independent manner, while FHA1 recognizes a phospho-epitope located in another region of the DDK complex. While this idea awaits validation, other dual interactions have been previously observed in the structures of other FHA domains, including that of KIF13B and Chk2 [52,54]. This seems to suggest that simultaneous phosphorylation dependent and independent interactions may be a broader mechanism to regulate interactions mediated by FHA domains than previously anticipated.…”

“…The first is with the ArfGAP domain of one of the CENTA1 molecules, which contacts the FHA loops that normally recognize a pThr. However, this interaction is phosphorylation-independent because the FHA domain of KIF13B lacks the conserved residues for phospho-threonine recognition [54]. The second CENTA1 molecule in the tetramer uses its Pleckstrin Homology 1 (PH1) domain to contact a surface on the β-sandwich of the same KIF13B FHA domain.…”

“…One member of this family, KIF13B, uses its FHA domain to transport PIP3-rich vesicles in order to facilitate axon development. This involves the formation of a tetrameric complex with CENTA1, which has been studied through X-ray crystallography [54]. This complex has two CENTA1 molecules and two kinesin molecules, with the FHA domain of each kinesin involved in two simultaneous interactions ( Figure 4).…”