Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
Department of Pediatrics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
Am J Hum Genet. 2018 Oct 4;103(4):579-591. doi: 10.1016/j.ajhg.2018.09.005.
p21-activated kinases (PAKs) are serine/threonine protein kinases acting as effectors of CDC42 and RAC, which are members of the RHO family of small GTPases. PAK1's kinase activity is autoinhibited by homodimerization, whereas CDC42 or RAC1 binding causes PAK1 activation by dimer dissociation. Major functions of the PAKs include actin cytoskeleton reorganization, for example regulation of the cellular protruding activity during cell spreading. We report the de novo PAK1 mutations c.392A>G (p.Tyr131Cys) and c.1286A>G (p.Tyr429Cys) in two unrelated subjects with developmental delay, secondary macrocephaly, seizures, and ataxic gait. We identified enhanced phosphorylation of the PAK1 targets JNK and AKT in fibroblasts of one subject and of c-JUN in those of both subjects compared with control subjects. In fibroblasts of the two affected individuals, we observed a trend toward enhanced PAK1 kinase activity. By using co-immunoprecipitation and size-exclusion chromatography, we observed a significantly reduced dimerization for both PAK1 mutants compared with wild-type PAK1. These data demonstrate that the two PAK1 variants function as activating alleles. In a cell spreading assay, subject-derived fibroblasts showed significant enrichment in cells occupied by filopodia. Interestingly, application of the PAK1 inhibitor FRAX486 completely reversed this cellular phenotype. Together, our data reveal that dominantly acting, gain-of-function PAK1 mutations cause a neurodevelopmental phenotype with increased head circumference, possibly by a combined effect of defective homodimerization and enhanced kinase activity of PAK1. This condition, along with the developmental disorders associated with RAC1 and CDC42 missense mutations, highlight the importance of RHO GTPase members and effectors in neuronal development.
p21 激活激酶（PAKs）是丝氨酸/苏氨酸蛋白激酶，作为 CDC42 和 RAC 的效应物起作用，CDC42 和 RAC 是 RHO 家族小 GTPases 的成员。PAK1 的激酶活性通过同源二聚体化被自身抑制，而 CDC42 或 RAC1 的结合导致 PAK1 通过二聚体解离而被激活。PAKs 的主要功能包括肌动蛋白细胞骨架的重排，例如在细胞扩展过程中调节细胞突出活性。我们报道了两个无关个体中 PAK1 突变 c.392A>G（p.Tyr131Cys）和 c.1286A>G（p.Tyr429Cys）的从头突变，这些个体表现为发育迟缓、继发的大头畸形、癫痫发作和共济失调步态。我们在一个供体的成纤维细胞中鉴定到 PAK1 靶标 JNK 和 AKT 的磷酸化增强，而在两个供体的成纤维细胞中鉴定到 c-JUN 的磷酸化增强，与对照供体相比。在两个受影响个体的成纤维细胞中，我们观察到 PAK1 激酶活性增强的趋势。通过共免疫沉淀和尺寸排阻色谱，我们观察到与野生型 PAK1 相比，两种 PAK1 突变体的二聚化显著减少。这些数据表明这两种 PAK1 变体作为激活等位基因发挥作用。在细胞扩展测定中，供体衍生的成纤维细胞显示出大量由丝状伪足占据的细胞富集。有趣的是，应用 PAK1 抑制剂 FRAX486 完全逆转了这种细胞表型。总的来说，我们的数据揭示了显性作用的、具有功能获得的 PAK1 突变导致神经发育表型，伴有头围增加，可能是由于 PAK1 同源二聚化缺陷和激酶活性增强的综合作用。这种情况，以及与 RAC1 和 CDC42 错义突变相关的发育障碍，突出了 RHO GTPase 成员和效应物在神经元发育中的重要性。
