Shapovalova Zoya, Tabunshchyk Kyrylo, Greer Peter A
Queen's University Cancer Research Institute, Division of Cancer Biology and Genetics, Kingston, Ontario, Canada.
BMC Dev Biol. 2007 Nov 30;7:133. doi: 10.1186/1471-213X-7-133.
Fps/Fes and Fer are the only two members of a distinct subclass of cytoplasmic protein tyrosine kinases. Fps/Fes was previously implicated in Semaphorin 3A (Sema3A)-induced growth cone collapse signaling in neurons from the dorsal root ganglion (DRG) through interaction with and phosphorylation of the Sema3A receptor component PlexinA1, and members of the collapsin response mediator protein (CRMP) family of microtubule regulators. However, the potential role of the closely related Fer kinase has not been examined.
Here we provide novel biochemical and genetic evidence that Fer plays a prominent role in microtubule regulation in DRG neurons in response to Sema3A. Although Fps/Fes and Fer were both expressed in neonatal brains and isolated DRGs, Fer was expressed at higher levels; and Fer, but not Fps/Fes kinase activity was detected in vivo. Fer also showed higher in vitro kinase activity toward tubulin, as an exogenous substrate; and this activity was higher when the kinases were isolated from perinatal relative to adult brain stages. CRMP2 was a substrate for both kinases in vitro, but both CRMP2 and PlexinA1 inhibited their autophosphorylation activities. Cultured mouse DRG neurons retracted their axons upon exposure to Sema3A, and this response was significantly diminished in Fer-deficient, but only slightly attenuated in Fps/Fes-deficient DRG neurons.
Fps/Fes and Fer are both capable of phosphorylating tubulin and the microtubule regulator CRMP2 in vitro; and their in vitro kinase activities were both inhibited by CRMP2 or PlexinA1, suggesting a possible regulatory interaction. Furthermore, Fer plays a more prominent role than Fps/Fes in regulating the axon retraction response to Sema3A in DRG neurons. Therefore, Fps/Fes and Fer may play important roles in developmental or regenerative axon pathfinding through signaling from Sema3A to the microtubule cytoskeleton.
Fps/Fes和Fer是细胞质蛋白酪氨酸激酶一个独特亚类中仅有的两个成员。Fps/Fes先前被认为通过与信号素3A（Sema3A）受体成分神经纤毛蛋白A1（PlexinA1）相互作用并使其磷酸化，以及与微管调节蛋白的塌陷反应介导蛋白（CRMP）家族成员相互作用，参与背根神经节（DRG）神经元中Sema3A诱导的生长锥塌陷信号传导。然而，与之密切相关的Fer激酶的潜在作用尚未得到研究。
在此，我们提供了新的生化和遗传学证据，表明Fer在DRG神经元对Sema3A的微管调节中起重要作用。尽管Fps/Fes和Fer在新生大脑和分离的DRG中均有表达，但Fer的表达水平更高；并且在体内检测到了Fer的激酶活性，而未检测到Fps/Fes的激酶活性。作为外源底物，Fer对微管蛋白也表现出更高的体外激酶活性；与成年脑阶段相比，从围产期脑分离的激酶的这种活性更高。体外实验中，CRMP2是这两种激酶的底物，但CRMP2和PlexinA1均抑制它们的自身磷酸化活性。培养的小鼠DRG神经元在暴露于Sema3A后会回缩其轴突，在Fer缺陷型DRG神经元中这种反应明显减弱，但在Fps/Fes缺陷型DRG神经元中仅略有减弱。
Fps/Fes和Fer在体外均能够使微管蛋白和微管调节蛋白CRMP2磷酸化；它们的体外激酶活性均受到CRMP2或PlexinA1的抑制，提示可能存在调节性相互作用。此外，在调节DRG神经元对Sema3A的轴突回缩反应中，Fer比Fps/Fes发挥更突出的作用。因此，Fps/Fes和Fer可能通过Sema3A向微管细胞骨架的信号传导，在发育或再生轴突导向中发挥重要作用。
