Caroline M. Dumas, Anna M. Schmoker, Shannon N. Bennett, Amara Chittenden, Chelsea Darwin, Helena Gaffney, H. Lewis, Eliana Moskovitz, Jonah Rehak, Anna Renzi, Claire Rothfelder, Adam Slamin, Megan Tammaro, L. Sweet, B. Ballif
{"title":"在Neuro2A细胞中鉴定的CRK和CRKL信号适配器SH2结构域的新相互作用体","authors":"Caroline M. Dumas, Anna M. Schmoker, Shannon N. Bennett, Amara Chittenden, Chelsea Darwin, Helena Gaffney, H. Lewis, Eliana Moskovitz, Jonah Rehak, Anna Renzi, Claire Rothfelder, Adam Slamin, Megan Tammaro, L. Sweet, B. Ballif","doi":"10.33697/ajur.2022.068","DOIUrl":null,"url":null,"abstract":"CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. 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This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. 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引用次数: 0
摘要
CT10激酶调控因子(CRK)和CRK样蛋白(CRKL)构成了一个信号转导蛋白家族,在多种细胞类型的基本细胞过程调控中发挥重要作用,包括细胞运动和增殖。CRK和CRKL的Src Homology 2 (SH2)结构域与含有磷酸化酪氨酸- x - x -脯氨酸(pYXXP)基序的蛋白相互作用,促进信号事件中复合物的形成。到目前为止,已经确定了少数CRK/ crkl - sh2特异性相互作用因子,尽管计算机分析表明仍有许多其他相互作用因子有待发现。为了鉴定可能参与神经元发育的CRK/CRKL-SH2相互作用物,我们使用神经细胞系(Neuro2A或N2A)进行了基于质谱的蛋白质组学筛选。结果鉴定出132个(已知6个,新发现126个)含有yxxp的CRK/CRKL-SH2相互作用物,其中77个在酪氨酸磷酸酶抑制后被刺激结合到CRK/CRKL-SH2结构域。大约一半的鉴定蛋白是CRK-和CRKL-SH2结构域的共同相互作用物。然而,两个CRK家族成员SH2结构域在实验重复中表现出独特的结合伙伴。这些发现揭示了大量新的神经元CRK/CRKL-SH2结构域结合伙伴,并表明CRK家族SH2结构域除了典型的pYXXP基元外,还具有未描述的对接偏好。关键词:CRK;CRKL;SH2;质/女士;蛋白质组学;神经发育;信号转导
Novel Interactors of the SH2 Domain of the Signaling Adaptors CRK and CRKL Identified in Neuro2A Cells
CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. KEYWORDS: CRK; CRKL; SH2; LC-MS/MS; Proteomics; Neurodevelopment; Signal Transduction