Mechanism of RIN1 Signaling in Neuronal Plasticity

描述(申请人提供)：RIN1蛋白是一种RAS效应器，主要在成熟的前脑神经元中表达。RIN1刺激ABL家族酪氨酸激酶，并指导细胞骨架重塑。RIN1缺失突变小鼠表现出显著的长时程增强(LTP)和记忆增强(即RIN1通常抑制学习)。H-、K-、NRAS和ABL2也在前脑神经元表达，并执行学习和记忆功能。这些研究为调节神经元可塑性(学习)的组织特异性RAS途径提供了关键的见解。了解RIN1的功能也可能对神经精神障碍有影响。RIN1与ABL蛋白结合。此外，RIN1作为ABL激酶的直接激活剂是独一无二的。RAS似乎刺激ABL2的RIN1激活，这三种蛋白(RAS、RIN1、ABL2)在体内被发现是一个复合体。这些数据证明了RAS和ABL蛋白之间的物理和功能联系。RIN1促进CRKII和CRKL的ABL2磷酸化，CRKII和CRKL是协调细胞骨架重构的接头蛋白。这项建议的第一个目的是确定RIN1刺激ABL2激酶活性的机制。这可能涉及底物特异性的增强(即RIN1为CRK蛋白提供了一个对接位置)或变构功能(即RIN1导致ABL2激酶活性位点的变化)。了解RIN1介导的刺激机制应该会扩大在学习和细胞转化中操纵ABL功能的机会。第二个目标是确定RIN1用来促进细胞骨架重塑的信号成分。RIN1和ABL2与CRKL在体内形成络合物。CRKII和CRKL通过多个SH2和SH3结合伙伴重组细胞骨架。识别RIN1/CRK(L)复杂成分将为刺激诱导和特定细胞类型的重塑提供新的见解。这项建议的第三个目的是评估RIN1对神经元可塑性的贡献。RIN1可以协调细胞骨架重塑，也可以与其他RAS效应器竞争。我们将使用培养的神经元和脑组织来确定神经元刺激如何参与RAS-RIN1-ABL2-CRK(L)通路，以及该通路的激活如何负面调节可塑性。将特别关注树突棘重塑，因为这一现象与学习的联系仍然是推测的。

DESCRIPTION (provided by applicant): The RIN1 protein is a RAS effector expressed predominantly in mature forebrain neurons. RIN1 stimulates ABL family tyrosine kinases and directs cytoskeletal remodeling. RIN1 null mutant mice show a striking increase in long term potentiation (LTP) and enhancement in memory (i.e. RIN1 normally inhibits learning). H-,K-, NRAS and ABL2 are also expressed in forebrain neurons and perform learning and memory functions. These studies provide critical insights into a tissue-specific RAS pathway that regulates neuronal plasticity (learning). Understanding RIN1 function may also have implications for neuropsychiatric disorders. RIN1 binds to ABL proteins. Moreover, RIN1 is unique as a direct ABL kinase activator. RAS appears to stimulate RIN1 activation of ABL2 and these three proteins (RAS, RIN1, ABL2) are found as a complex in vivo. These data demonstrate a physical and functional connection between RAS and ABL proteins. RIN1 promotes the ABL2 phosphorylation of CRKII and CRKL, adaptor proteins that coordinate cytoskeletal remodeling. The first aim of this proposal is to define the mechanism by which RIN1 stimulates ABL2 kinase activity. This may involve a substrate-specific enhancement (i.e. RIN1 provides a docking site for CRK proteins) or an allosteric function (i.e., RIN1 induces changes in the ABL2 kinase active site). Understanding the RIN1- mediated stimulation mechanism should expand opportunities for manipulating ABL function in learning, as well as in cell transformation. The second aim is to define signaling components used by RIN1 to promote cytoskeletal remodeling. RIN1 and ABL2 are complexed with CRKL in vivo. CRKII and CRKL reorganize the cytoskeleton through multiple SH2 and SH3 binding partners. Identification of RIN1/CRK(L) complex components will provide new insights into stimulus-induced and cell type specific remodeling. The third aim of this proposal is to evaluate the contribution of RIN1 to neuronal plasticity. RIN1 can coordinate cytoskeletal remodeling as well as compete with other RAS effectors. We will use cultured neurons and brain tissue to determine how neuronal stimulation engages the RAS-RIN1-ABL2-CRK(L) pathway and how activation of this pathway negatively regulates plasticity. Special focus will be placed on dendritic spine remodeling because the connection of this phenomenon to learning remains conjectural.