Control of a kinesin-cargo linkage mechanism by JNK pathway kinases

Results and Discussion

Maintaining proper distributions of protein complexes, RNAs, vesicles, and other organelles in axons is critical for the development, function, and survival of neurons. The primary distribution mechanism relies on long-distance transport driven by microtubule motor proteins [2]. Components newly synthesized in the cell body, but needed in the axon, bind kinesin motors that carry them toward microtubule plus-ends and the axon terminal (anterograde transport). Components in the axon that require transport to the cell body, such as neurotrophic signals and endosomes, bind dynein motors that carry them toward minus ends (retrograde transport). The importance of these processes is highlighted by the observation that mutation of motors and other transport machinery components can cause neurodegenerative diseases in humans and analogous phenotypes in model organisms [1, 7, 8].

Two key questions are 1) how do cargoes link to particular motors? and 2) how are such linkages regulated to ensure appropriate pick-up and drop-off dynamics? For kinesin-vesicle linkages, scaffolding proteins have emerged as key connectors. For example, the cargo-binding kinesin light chain (Klc) subunit of kinesin-1 binds not only the kinesin-1 heavy chain (Khc), but also JNK interacting proteins (JIPs) [4, 5, 9]. Vertebrate JIPs can bind multiple components of the JNK signaling pathway, e.g. JNK itself, upstream activating kinases (MAPKKs), and regulatory kinases (MAPKKKs) [5, 6]. JIPs can also bind vesicle-associated membrane proteins, such as ApoER2, which is a reelin receptor, and APP, a key factor in Alzheimer's disease [4, 10, 11]. Therefore, JIP scaffolding proteins likely link JNK pathway kinases and kinesin-1 to vesicles carrying these membrane proteins. This raises an interesting question: Are the JNK pathway kinases simply passive hitchhikers on the kinesin-1/JIP/vesicle complex, or can they actively regulate its transport [12]?

We conducted a genetic screen for factors that control kinesin-JIP linkage during axonal transport. The screen was based on the previous observation that neuron specific overexpression of Aplip1, which encodes the Drosophila JIP1, causes synaptic protein accumulation in axons, larval paralysis, and larval-pupal lethality [13], which are the classic axonal transport disruption phenotypes caused by Khc and Klc mutations [8, 14]. Why might overexpression of the JIP1 cargo linker for kinesin-1 disrupt axonal transport? The disruptive effect requires APLIP1 (JIP1)-Klc binding [13]. It may be that excess APLIP1 (JIP1) competes with other Klc-binding proteins; for example, different linkers that may attach kinesin-1 to other cargoes. In search of factors that can disrupt or antagonize APLIP1 (JIP1)-Klc binding, we screened for genes that can suppress the axonal transport phenotypes when co-overexpressed with Aplip1. We screened an “EP” collection of fly strains capable of the targeted overexpression of endogenous Drosophila genes [15, 16] and identified P{EP}faf^EP381, a line that overexpresses fat facets (faf) [17], as a strong suppressor of the APLIP1 (JIP1)-induced lethality and other neuronal overexpression phenotypes (Fig. 1A to C, and Table S1 in Supplemental Data, available online).

Faf protein antagonizes ubiquitination and proteasome-mediated degradation of its target proteins [18, 19]. Interestingly, Faf was recently reported to stimulate a Drosophila neuronal JNK signaling pathway that is regulated by the MAPKKK Wallenda (Wnd) [20], a homolog of dual leucine zipper-bearing kinase (DLK) which is known to bind JIP1 [6, 21]. Overexpression of faf leads to increased levels of Wnd (MAPKKK) protein, which causes excessive synaptic sprouting through a pathway that requires the Drosophila JNK homolog Basket (Bsk) [20]. We found that mutating just one copy of wnd blocked the suppression of Aplip1 overexpression by P{EP}faf^EP381 (Fig. 1D and Table S1). This suggests that faf overexpression suppresses APLIP1 (JIP1)-Klc interaction by elevating the level of Wnd (MAPKKK). Consistent with this, direct overexpression of wnd in neurons using a wild-type transgene (UAS-wnd) was as effective as P{EP}faf^EP381 in suppressing UAS-Aplip1-induced axonal accumulation of synaptic proteins (Fig. 1F, Table S1). Equivalent expression of a “kinase-dead” mutant transgene (UAS-wnd^KD) did not suppress the defects (Fig. 1G, Table S1). Thus, Wnd (MAPKKK) and its downstream phosphorylation targets may actively regulate APLIP1 (JIP1)-Klc binding in neurons.

If Wnd (MAPKKK) signaling plays a role in normal axonal transport, disrupting its function should cause axonal transport phenotypes. Consistent with this, wnd loss-of-function mutations (wnd¹/wnd²) in an otherwise wild-type background caused accumulation of synaptic proteins in axons (Fig. 2B, Table S1). The accumulation phenotype was rescued by motoneuron expression of the wild-type wnd transgene, but not by equivalent expression of the kinase-dead mutant transgene (Fig. 2C and D, Table S1). The likely target of Wnd (MAPKKK) kinase activity is the Drosophila homolog of MKK7, Hemipterous (Hep), a MAPKK that activates Bsk (JNK) [22, 23]. Mutation of hep also caused axonal accumulations, as did neuronal expression of a dominant-negative mutant bsk transgene (Fig. 2E and F, Table S1). The results of these genetic inhibition tests combined with those of the Aplip1 overexpression suppression tests suggest that a Wnd (MAPKKK)-activated JNK pathway influences fast axonal transport by regulating APLIP1 (JIP1)-Klc binding.

Is a Wnd (MAPKKK)-Hep (MAPKK)-Bsk (JNK) signaling module bound by APLIP1 (JIP1)? While all three components of the homologous vertebrate module (DLK-MKK7-JNK) bind JIP1 [5, 6], APLIP1 (JIP1) lacks a conserved JNK binding domain, and it does not bind directly to Bsk (JNK) [3]. However, APLIP1 (JIP1) does bind Hep (MAPKK), Klc, and the Drosophila APP homolog APPL [3]. To determine if Wnd (MAPKKK) associates with Hep (MAPKK) and/or APLIP1 (JIP1), we performed co-expression and immunoprecipitation tests in Drosophila S2 cultured cells. Wnd (MAPKKK) did not co-precipitate with APLIP1 (JIP1) (Fig. 3A). However, Hep (MAPKK) did co-precipitate with APLIP1 (JIP1), and Wnd (MAPKKK) coprecipitated with Hep (MAPKK) (Fig. 3B and C). Thus, Wnd (MAPKKK) may bind and influence the APLIP1 (JIP1)-kinesin complex via Hep (MAPKK).

Can Wnd (MAPKKK) and Hep (MAPKK) control the binding of APLIP1 (JIP1) to Klc? When expressed in S2 cells, APLIP1 (JIP1) and Klc exhibit strong binding, as assessed by co-immunoprecipitation (Fig. 3D, lane 1) [13]. Co-expression of wild-type Wnd (MAPKKK) partially inhibited that APLIP1 (JIP1)-Klc binding, but co-expression of a kinase-dead mutant Wnd (MAPKKK) did not (Figure 3D, lanes 2 and 3). Wild-type Hep (MAPKK) also caused a partial inhibition of APLIP1 (JIP1)-Klc binding, and a constitutively active mutant Hep (MAPKK) caused nearly complete inhibition (Fig. 3D, lanes 4 and 5). Finally, co-expression of wild-type Wnd (MAPKKK) and Hep (MAPKK) together caused an almost complete inhibition of APLIP1 (JIP1)-Klc binding (Fig. 3D, lane 6). In addition to inhibiting APLIP1 (JIP1)-Klc binding, Wnd-Hep activation in S2 cells increased the level of Bsk (JNK) activation (Fig. 3D). Hence, there is a correlation between decreased levels of APLIP1 (JIP1)-Klc binding and elevated levels of Bsk (JNK) activation. This suggests that, despite the lack of a known JNK binding site on APLIP1 (JIP1), Bsk (JNK) may be the kinase that disrupts the APLIP1 (JIP1)-Klc complex. These results suggest that Wnd (MAPKKK) activation of Hep (MAPKK), and perhaps also Hep (MAPKK) activation of Bsk (JNK), can regulate the linkage between kinesin-1 and a cargo complex via the JIP1-like scaffolding protein, APLIP1 (Fig. 4).

Hep (MAPKK) may regulate the APLIP1 (JIP1) complex either by activating JNK (Fig. 4, pathway 1) or by a mechanism independent of JNK (Fig. 4, pathway 2). Our observations that motoneuron specific inhibition of Bsk (JNK) caused transport defects similar to those caused by mutations in wnd and hep (Fig. 2B, E and F) and that decreased APLIP1 (JIP1)-Klc binding in S2 cell lysates coincided with increased phosphorylated Bsk (JNK) (Fig. 3D) support pathway 1, i.e. Hep (MAPKK) activation of Bsk (JNK), which then directly or indirectly inhibits APLIP1 (JIP1)-Klc binding. Pathway 2 employs an alternative mechanism in which activated Hep (MAPKK) does not need Bsk (JNK) to inhibit APLIP1 (JIP1)-Klc binding. There is little current evidence that Hep or its vertebrate MAPKK homolog MKK7 have phosphorylation targets other than Bsk (JNK) [22, 24]. However, that does not exclude the possibility that activated Hep propagates a direct conformational change to APLIP1 (JIP1) that causes Klc dissociation. Regardless of how Hep (MAPKK) disrupts binding, when kinesin-1 is not attached to cargo via JIP1, it can fold into a compact form that does not interact with microtubules [25]. Hence the activated Wnd (MAPKKK) pathway could both inhibit APLIP1 (JIP1)-Klc binding and it could cause dissociation of kinesin-1 from microtubules (Fig. 4). Consistent with this, recent studies report that stimulation of JNK pathways in cultured cells or axoplasm can disrupt the association of kinesin-1 with microtubules [26, 27].

From a broader perspective on axonal transport regulation, it is interesting to consider that there are multiple types of kinesin-1 cargoes [2], there are various JIPs that could be specific for different cargoes [4, 5, 9], and different MAPKKKs can associate with different JIPs [5, 6]. By sitting at the top of a classic signaling cascade, MAPKKKs like Wnd are in a good position to differentially control the transport of specific subsets of anterograde kinesin-1 cargoes in response to specific cellular signals. It is known in mammals that other MAPKKKs such as MLK, ASK1, and MEKK1 can bind JIP scaffolding proteins [28]. It will be interesting to determine if they too influence kinesin-cargo interactions.

To our knowledge, the work presented here provides the first demonstration that a kinesin and its transport functions can be influenced by a MAPKKK. More specifically, the MAPKKK Wnd and its downstream MAPKK Hep can regulate attachment of a JIP1 cargo linker to kinesin-1. Our results also provide the first indication that ubiquitination pathways, by way of MAPKKKs, could be important for proper regulation of axonal transport. Finally, our results suggest that JNK pathway kinases are not just hitchhikers on the axonal kinesin-1/JIP/cargo complex, rather they can actively regulate its transport dynamics.

Supplementary Material