Here we show that Wnt-Dvl signalling regulates MT dynamics through two distinct pathways. Dvl induces the concomitant inhibition of Gsk3β and the activation of JNK to increase MT stability. The typical PCP pathway appears not to be involved as small Rho GTPases are not required for this function. These findings highlight a new role for JNK kinase in the modulation of the MT dynamics mediated by Wnt signaling.
We have previously showed that a divergent canonical Wnt pathway regulates MT dynamics. The scaffold protein Dvl is tightly associated with MTs [4
] and increases MT stability through the local inhibition of a pool of Gsk3β [5
]. In turn, inhibition of Gsk3β by Wnt or expression of Dvl leads to changes in the phosphorylation of MAPs, notably MAP1B, resulting in increased MT stability. Although pharmacological inhibition of Gsk3β increases MT stability, this effect is weaker than that observed by expression of Dvl. Moreover, expression of Gsk3β only partially blocks the ability of Dvl to stabilize MTs [5
]. These findings suggest that in addition to Gsk3β, Wnt-Dvl signals through another pathway to regulate MT dynamics.
The family of the small Rho GTPases are not required for the regulation of MT stability upon Wnt activation. Although small Rho GTPases have been extensively shown to control different aspects of actin dynamic and organisation [18
], these molecules can also regulate the MT network. For example RhoA and its effector mDia are necessary for MT stability induced by LPA and activation of integrins [16
]. Conversely Rac and Cdc42 signalling through their effectors PAK and IQGAP respectively are required for the capture of MT plus-end at the leading edge of migrating cells [27
] and for inhibition of the MT destabilising effect of Stathmin (OP18) [17
]. Recently Cdc42 and RhoA have been shown to regulate the phosphorylation of Gsk3β to control MT stability and cell polarisation [28
]. Importantly, the PCP pathway activates small Rho GTPases to regulate tissue polarity, cell migration and dendritic morphogenesis, processes that require the reorganization of the cytoskeleton [26
]. However, expression of Rho, Rac or Cdc42 dominant-negative mutants does not alter the ability of Dvl to stabilise MTs. Thus, Wnt-Dvl signalling regulates MT stability through a Rho GTPase independent pathway.
Wnt signalling regulates MT stability through JNK. In neurons, activation of JNK increases MT stability whereas pharmacological inhibition of JNK or expression of a dominant-negative form of JNK blocks the effect of Dvl on MT stability. In the PCP pathway, JNK is downstream of Rho GTPases [30
]. However, the finding that small GTPases are not required for MT stabilization by Dvl suggests that activation of JNK is achieved independently of these small GTPases. Interestingly, studies have shown that Dvl can activate JNK in a Rac and Cdc42 independent manner [32
]. This is the first demonstration that Wnt signalling regulates MT stability through JNK.
The same domains of Dvl are required for signalling to JNK and Gsk3β to modulate MT dynamics. We have previously shown that the PDZ and DIX domains of Dvl are important for signalling through Gsk3β [4
]. Here we show that the PDZ is required for binding of Dvl to the MT network. Moreover, the PDZ and DIX domains are also required for Dvl to signal to JNK. It has been generally accepted that PDZ domain of Dvl is required for the activation of the Gsk3β/β-catenin pathway whereas the DEP domain is required for the PCP pathway [37
]. However, several studies have shown that the PDZ domain of Dvl is also required for the PCP pathway [26
] suggesting that different domains of Dvl can activate different Wnt signalling pathways depending on the cellular or developmental context. Thus, our finding that Dvl signals to JNK through its PDZ and DIX domains raises the possibility that Gsk3β and JNK act together to regulate MT dynamics.
How does JNK modulate MT dynamics? JNK could regulate MT stability through changes in gene expression or through direct signalling to the cytoskeleton. JNK has been well established as a key regulator of transcription [49
]. However, we found that Wnt regulates MT stability through a transcription-independent pathway [5
] suggesting that the Wnt pathway through JNK directly signals to the cytoskeleton. Consistently, we found that Dvl increases the level of active JNK associated with MTs suggesting that JNK can directly modulate the MT network [51
] by changing the phosphorylation of MT associated proteins such as MAP2 and MAP1B [29
]. Moreover, Wnt signalling regulates the phosphorylation of MAPs suggesting that JNK could mediate these changes. Future studies will elucidate the targets of JNK in this novel Wnt pathway.
Two different pathways that include JNK and Gsk3β contribute to Wnt-mediated effects on MT dynamics. Epistatic analyses show that the concomitant expression of Gsk3β and pharmacological inhibition of JNK strongly blocks the ability of Dvl to stabilize MTs. Interestingly, activation of Gsk3β or inhibition of JNK alone partially blocks Dvl function. These findings indicate that these two kinases are both required to regulate MTs stability via Dvl. Interestingly JNK is not downstream of Gsk3β as activation of JNK does not alter the ability of Gsk3β to block MT stability induced by Dvl. Moreover when JNK is blocked, Dvl is still able to inhibit Gsk3β activity as determined by the level of MAP1B phosphorylation. Conversely when Gsk3β is expressed in neurons the activity of JNK is not altered. Taken together these findings suggest that JNK regulates the cytoskeleton through a distinct pathway that is independent of Gsk3β. In summary, our studies demonstrate a novel role for JNK in Wnt-mediated MT stability and demonstrate that Wnts can simultaneously active different signalling branches to modulate complex cellular processes.