In this study, we identified several key signaling molecules and cellular processes required for endogenous EphB2-mediated cortical neuron GCC (), including a novel role for Pak in this process. We report a novel interaction between kinase-active EphB2 and Pak1, which likely occurs via interaction with the EphB2-interacting adaptor protein, Nck, and we show that Pak kinase activity and Nck are required for ephrinB2-induced GCC. However, our data suggest that ephrinB2 binding to EphB2 does not elevate Rac1-GTP to levels detectibly above baseline, which is consistent with a lack of effect by Rac/Cdc42 dominant negative expression on EphB2-mediated GCC. We also provide evidence that Pak1’s function in EphB2-dependent GCC requires binding to Nck, Pak kinase activity, and residues that are known to mediate binding to the PIX/COOL GEFs. However, a direct interaction between Pak1 and Rac/Cdc42-GTP does not appear to be required. These data suggest that recruitment of Pak to the ephrinB2/EphB2 receptor complex is necessary and sufficient to mediate its role in GCC. Consistent with previous studies, RhoA-GTP, Rho Kinase and endocytosis are all required for GCC, suggesting that the ephrinB2/EphB2 complexes recruit multiple key signaling components to orchestrate growth cone F-actin remodeling during repulsive guidance.
While our findings reveal a novel role for Pak signaling in EphB2-mediated GCC, these findings also raise many new questions for future study. Classically, Pak is thought to be an effector of Rac/Cdc42-GTP, and subsequent enhancement of Pak kinase activity results in F-actin remodeling through sequential activation of Lim kinase and phosphorylation of the F-actin binding protein, cofilin (Edwards et al., 1999
). However, this pathway often results in generation of lamellapodia, membrane ruffles and focal adhesions (reviewed in (Bamburg, 1999
)), which is in apparent conflict with presumed dynamics in growth cone repulsion. In our study, we did not observe any evidence that ephrinB2 treatment stimulates this Rac/Pak/LimK signaling cascade, as we do not observe an increase P-cofilin levels in response to ephrinB2 ( & Supplementary Figure 2
). Rather, we now report an important role for Pak1 and Nck in ephrinB2-induced GCC largely independent of Rac/Cdc42-GTP, which suggests that recruitment of Pak1/Nck to the activated EphB2 receptor complex has a distinct cellular function than Pak activated in response to upregulated Rac/Cdc42-GTP.
Previous work has reported a critical role for drosophila Pak and the Nck homolog, Dock, in the process of photoreceptor axon guidance in vivo
(Garrity et al., 1996
; Hing et al., 1999
). In loss-of-function Dock or Pak mutant flies, R photoreceptor cell axons are mistargeted and disorganized within the lamina and medulla of the brain. Rescue studies in vivo
revealed a critical role for Pak kinase activity, binding to PIX and Dock, and Rac/Cdc42-GTP binding (Hing et al., 1999
). However, in the fruit fly, Rac/Cdc42 binding to Pak is required for R photoreceptor cell axon guidance (Hing et al., 1999
), and as such, represents a significant departure from the mechanisms of EphB2-mediated cortical GCC in rodents. Interestingly, in both flies and mammals, the recruitment of Pak to the cell membrane appears to be sufficient to activate Pak kinase activity (Hing et al., 1999
; Lu et al., 1997
), so it is possible that Nck-mediated recruitment of Pak1 to the activated EphB2 receptor at the cell surface increases its intrinsic kinase activity, but Pak1 autophosphorylation of the S/T sites only occurs upon Rac/Cdc42-GTP binding. As such, we speculate that either: (1) the basal Pak kinase activity, recruited to the EphB2 receptors, is sufficient for its role in GCC or (2) the recruitment of Pak to the activated EphB2 receptors at the plasma membrane increases Pak1 kinase activity in a Rac-independent manner. We observed that Pak1 isolated from ephrinB2-treated neurons possesses increased kinase activity toward a known substrate, MBP (Supplemental Figure S2
), suggesting that Pak activity has indeed been altered in response to ephrinB2 treatment.
Nck genes are critical for proper cortical axon guidance in vivo
such that the conditional deletion of both Nck1 and Nck2 genes leads to deficits in corticospinal tract axon guidance and a reduced posterior anterior commissure (Fawcett et al., 2007
). Interestingly, individual Nck1 or Nck2 KO mice appeared normal, suggesting that these two genes serve largely redundant functions during development or can compensate for the loss of the other gene. Similar to this study, we found that expression of the Nck2 (ΔSH3-2) mutant also blocked ephrinB2-induced GCC (N. Srivastava, unpublished observations), suggesting functional redundancy in EphB2-mediated GCC in vitro
. Interestingly, Nck2 recruits Pak1 to mediate ephrinB3 reverse signaling-dependent axonal pruning of hippocampal dentate gyrus neurons during development (Xu and Henkemeyer, 2009
), suggesting that Nck/Pak may be a common signaling complex for both forward (Eph receptor) and reverse (ephrinB) repulsive axon guidance signaling.
In addition to ephrin-induced recruitment of signaling proteins to the clustered Eph receptors, ephrin-induced endocytosis has emerged as an important step for forward signaling and GCC (Cowan et al., 2005
; Fournier et al., 2000
; Jurney et al., 2002
; Marston et al., 2003
; Zimmer et al., 2003
). Our present findings that both dynamin (K44A) mutant and PAO block ephrinB2-induced GCC () are consistent with a key role for EphB2 and/or plasma membrane internalization in GCC. Surprisingly, we do not observe a key role for Rac-GTP in ephrinB2-induced GCC, which is distinct from findings of ephrinB2/EphB4-mediated cell-cell repulsion in a Swiss 3T3 cultures (Marston et al., 2003
). However, it is interesting to note that Pak1 kinase activity, independent of Rac/Cdc42 binding, has been linked to a form of endocytosis, macropinocytosis, in non-neuronal cells (Dharmawardhane et al., 2000
). It is admittedly difficult to interpret a negative result from the Rac dominant negative experiments in our study, but taken together with the absence of ephrinB2-induced Rac-GTP and P-Pak production (), the failure of the Rac/Cdc42-binding mutant of Pak (H83L,H86L) to reduce ephrinB2-induced GCC (), and the failure of the NSC “Rac inhibitor” to block GCC, these combined findings suggest that Rac-GTP function is likely dispensable for Pak’s role in ephrinB2-induced GCC in this context.
Taken together, our findings reveal important EphB2 receptor forward signaling mechanisms that are required for ephrinB2-induced cortical growth cone collapse. Considering the importance of axon guidance for proper cortical neuronal connectivity in vivo, the discovery of new repulsive signaling factors, such as Nck and Pak, may reveal new therapeutic targets for the treatment of neurodevelopmental disorders or axonal regeneration following brain injury.