Here we have shown that GAKIN/KIF13B plays a critical role in axon formation of hippocampal neurons, which is regulated by Par1b-mediated phosphorylation. The first mammalian counterpart of Par1 discovered was MARK, a kinase that destabilizes microtubules (10
). The effect of Par1 on microtubules has generally been attributed to its ability to phosphorylate several MAPs, including Tau and MAP2. Whether such MAPs are involved directly in regulation of polarity is unknown. In this regard, it should be noted that accumulation of GAKIN/KIF13B in the longest neurite and determination of polarity occur at the same time (at the transition from stage 2 to 3 [Fig. ]). Furthermore, overexpression of GAKIN/KIF13B could partially induce extra axon formation (Fig. ), and knockdown analyses supported the involvement of GAKIN/KIF13B in axon specification (Fig. ). Taken together with the previous report (14
), GAKIN/KIF13B appears to be an important regulator of neuronal polarity.
In addition, GAKIN/KIF13B is the mammalian ortholog of Drosophila
Khc-73, which has been shown to play a crucial role in microtubule-induced Pins/Gαi cortical polarity (32
). The two phosphorylation sites in GAKIN/KIF13B are also conserved in Khc-73 (Fig. ), suggesting that GAKIN might be an important player for cell polarity, of which activity is regulated by phosphorylation. However, in the regulation of Pins/Gαi cortical polarity, Khc-73 functions in a manner independent of Par proteins, and thus, the regulatory mechanism of GAKIN/KIF13B by Par1b in this study cannot be simply applicable to the understanding of the Drosophila
Pins/Gαi cortical polarity. This suggests an interesting possibility that some unidentified kinase(s) might phosphorylate and regulate Khc-73.
To further understand the role of Par1b in axon specification, it is very important to clarify the spatiotemporal activation/inactivation pattern of Par1b. Chen et al. (7
) utilized anti-phospho-Par1b antibody to detect inactivated Par1b, because it is generally thought that Par1b phosphorylation by aPKC leads to its functional inactivation. The authors indicated that phosphorylated Par1b accumulates at the tips of axons, suggesting that Par1b is inactivated in a single neurite, probably by the function of the PI3K and Par complex, and this neurite would then become an axon. Therefore, Par1b inactivation in one neurite appears to be a key step for the specification of axon from multiple candidate neurites. Because Par1b inhibits the accumulation of GAKIN/KIF13B at the tips of neurites (Fig. ), GAKIN/KIF13B accumulation should be allowed to occur in a single Par1b-inactivated neurite and stimulate it to become an axon.
Both endogenous and overexpressed GAKIN/KIF13B tend to accumulate at the tips of protrusions, but how such accumulation occurs is unknown. We found that a GAKIN/KIF13B mutant lacking the N-terminal Motor domain partly localized at the centrosome (data not shown), suggesting that GAKIN/KIF13B first localizes at the centrosome and then moves on microtubules to the tips of neurites or axons. When GAKIN/KIF13B reaches the distal ends of microtubules, it would become docked and accumulate there. The results shown in Fig. S3B in the supplemental material indicate that the Par1b-mediated phosphorylation inhibits the interaction of GAKIN/KIF13B with microtubules. This would be important for Par1b to inhibit GAKIN accumulation, because phosphorylated GAKIN/KIF13B loses the microtubule-binding ability and cannot move on microtubules.
kinase assays indicated that Par1b phosphorylates GAKIN/KIF13B at Ser1381 and Ser1410 (Fig. ). Ser1381 matches the consensus for the 14-3-3 binding motif, and indeed, 14-3-3 binding with GAKIN was confirmed by coimmunoprecipitation analyses (Fig. ). Monomeric 14-3-3 directly binds one phosphorylated Ser in its target protein, but it functions as a dimer through simultaneous binding to another phosphorylation site, which promotes stable association with the target (40
). Ser1410 might be the secondary phosphorylation site for 14-3-3, because the S1410A mutation did not abolish but slightly reduced the amount of coimmunoprecipitation (Fig. ). The results shown in Fig. S6 in the supplemental material suggest the negative role of 14-3-3 in axon formation, but the amount of endogenous 14-3-3β appeared to be very small or absent at the tips of not only axons but also of minor neurites. When GAKIN/KIF13B is phosphorylated, it should associate with 14-3-3 and dissociate from the microtubules. Therefore, GAKIN/KIF13B is not able to accumulate at the tips of minor neurites by utilizing the microtubules, which might explain the reason why 14-3-3 inhibits the elongation of minor neurites even though it does not exist at the tips.
It remains unresolved how GAKIN/KIF13B accumulation contributes to axon formation. GAKIN/KIF13B is a motor protein that can transport PIP3-containing lipid vesicles on microtubules through an interaction with centaurin-α1/PIP3BP (14
). Because PIP3 is known to accumulate at axon tips and plays an essential role in axon specification (23
), GAKIN/KIF13B might contribute to axon formation through regulation of PIP3 accumulation at axon tips. On the other hand, our experimental results with the PI3K inhibitor placed PIP3 upstream of GAKIN/KIF13B (Fig. ). Therefore, GAKIN/KIF13B can be placed both upstream and downstream of PIP3.
One possible scenario is that GAKIN/KIF13B might participate in a positive-feedback pathway that spatially regulates PIP3 (Fig. ). A similar feedback regulatory mechanism of PIP3 was first discovered in chemoattractant- or PIP3-stimulated neutrophils (37
). In this feedback loop, PI3K and small GTPases in the Rho family, including Rho, Rac, and Cdc42, play crucial roles. In the axon specification process, the Par complex mediates Cdc42-induced Rac activation (26
), and this mechanism is thought to play a role in positive-feedback regulation of PIP3, because activated Rac can bind PI3K (35
). Such feedback mechanisms should play essential roles in polarity formation, especially spontaneous polarity formation (2
) as is observed in the axon specification from several candidate neurites.
FIG. 8. Schematic diagram of a model for positive-feedback signaling mediated by Par1/GAKIN/KIF13B. PIP3 is generated from PIP2 by PI3K and activates aPKC via PDK1. Activated aPKC forms the complex with Par3/Par6 and phosphorylates Par1. Phosphorylated Par1 is (more ...)
GAKIN/KIF13B localization is dependent not only on PIP3 but also on the integrity of microtubules because nocodazole treatment abolishes GAKIN/KIF13B localization (see Fig. S3A in the supplemental material). Such behavior is similar to those of cytoplasmic linker protein (CLIP)-associated proteins (CLASPs). CLASPs are microtubule plus-end-tracking proteins (+TIPs) that can interact with other +TIPs, such as CLIPs and end-binding protein 1 (EB1) (1
). CLASPs also localize at the distal ends of microtubules in the leading edge of serum-stimulated motile fibroblasts, which was inhibited by the treatment with PI3K inhibitors. It was later reported that CLASPs associate directly with LL5β, a pleckstrin homology (PH) domain-containing protein that can bind to PIP3 (27
) and accumulate at the cortical areas (20
). In addition, LL5β is localized around focal adhesions where the cell adheres to the extracellular matrix through integrins. It would be interesting to examine the importance of GAKIN/KIF13B in cellular processes, such as motility and adhesion, and clarify the functional relationship between GAKIN/KIF13B and other PIP3-regulated +TIPs.