Winkler Sabine C, Shimobayashi Etsuko, Kapfhammer Josef P
Anatomical Institute, Department of Biomedicine, University of Basel, Pestalozzistrasse 20, CH - 4056, Basel, Switzerland.
Mol Neurobiol. 2020 Dec;57(12):5150-5166. doi: 10.1007/s12035-020-02038-6. Epub 2020 Aug 29.
The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.
信号蛋白PKCγ是浦肯野细胞发育和突触功能的主要调节因子。我们之前已经表明，PKCγ活性增加会损害小脑浦肯野细胞的树突发育。蛋白激酶Cγ基因（PRKCG）的突变会导致14型脊髓小脑共济失调（SCA14）。在表达人类S361G突变的SCA14转基因小鼠模型中，小脑切片培养物中浦肯野细胞的树突发育受损，类似于PKC的药理学激活。PKCγ驱动的树突生长抑制机制仍不清楚。通过免疫沉淀耦合质谱分析，我们确定了塌陷反应介导蛋白2（CRMP2）是一种与组成型活性PKCγ（S361G）相互作用的蛋白，并通过Duolink™邻近连接分析证实了这种相互作用。我们表明，在来自PKCγ（S361G）小鼠的小脑切片培养物中，浦肯野细胞中已知的PKC靶位点Thr555处的CRMP2磷酸化增加，证实了PKCγ对CRMP2的磷酸化。miRNA介导的CRMP2敲低减少了解离的小脑培养物中浦肯野细胞的树突生长，用修饰的Thr555位点转染CRMP2突变体也有同样的效果。相反，野生型CRMP2过表达后树突发育正常。在一种仅表达磷酸化缺陷型T555A突变体CRMP2的新型敲入小鼠中，解离培养物中浦肯野细胞的树突发育减少。这种减少可以通过转染野生型CRMP2来挽救，但只能部分地通过磷酸化模拟T555D突变体来挽救。我们的研究结果确定CRMP2是浦肯野细胞中PKCγ磷酸化的重要靶点，介导其对树突发育的控制。通过PKCγ对CRMP2磷酸化的动态调节是其正确功能所必需的。
