目的:蛋白激酶C(PKC)家族的常规成员,包括PKCβII,在三个主要基序上组成性磷酸化,并以引发状态位于胞质溶胶中。为了响应细胞刺激,PKCβII通过诱导型磷酸化和Mdm2介导的泛素化被激活。在这项研究中,我们的目的是确定PKCβII的激活机制,专注于调节磷酸化和泛素化的信号级联。
方法:使用显示不同调节特性的PDK1/PKCβII的功能丧失方法和突变体来鉴定负责胞吞的细胞成分和过程。
结果:Phorbol12-肉豆蔻酸酯13-乙酸酯(PMA)诱导PKCβII的磷酸化和泛素化,这是它易位到质膜所需要的,需要同时存在Gβγ和14-3-3ε。Gβγ和14-3-3ε通过支架PI3K和PDK1在细胞质中介导PKCβII的组成型磷酸化,这是一个不活跃的,但需要通过后续信号激活PKCβII的状态。为了应对PMA治疗,信号复合物转位到细胞核,PI3K与细胞核分离。此后,PDK1与14-3-3ε稳定相互作用并去磷酸化;PKCβII与Mdm2和Gβγ相互作用,导致其在C尾的两个赖氨酸残基上的泛素化。最后,PDK1/14-3-3ε和泛素化PKCβII转位至质膜。
结论:由于PKCβII介导广泛的细胞功能,在各种疾病的发病机制中起重要作用,我们的结果将为了解PKCβII相关疾病的发病机制和促进其治疗提供线索。
OBJECTIVE: Conventional members of protein kinase C (PKC) family, including PKCβII, are constitutively phosphorylated on three major motifs and located in the cytosol in a primed state. In response to cellular stimuli, PKCβII is activated through inducible phosphorylation and Mdm2-mediated ubiquitination. In this study, we aimed to identify the activation mechanism of PKCβII, focusing on the signaling cascade that regulate the phosphorylation and ubiquitination.
METHODS: Loss-of-function approaches and mutants of PDK1/PKCβII that display different regulatory properties were used to identify the cellular components and processes responsible for endocytosis.
RESULTS: Phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation and ubiquitination of PKCβII, which are needed for its translocation to the plasma membrane, required the presence of both
Gβγ and 14-3-3ε.
Gβγ and 14-3-3ε mediated the constitutive phosphorylation of PKCβII by scaffolding PI3K and PDK1 in the cytosol, which is an inactive but required state for the activation of PKCβII by subsequent signals. In response to PMA treatment, the signaling complex translocated to the nucleus with dissociation of PI3K from it. Thereafter, PDK1 stably interacted with 14-3-3ε and was dephosphorylated; PKCβII interacted with Mdm2 along with
Gβγ, leading to its ubiquitination at two lysine residues on its C-tail. Finally, PDK1/14-3-3ε and ubiquitinated PKCβII translocated to the plasma membrane.
CONCLUSIONS: As PKCβII mediates a wide range of cellular functions and plays important roles in the pathogenesis of various diseases, our results will provide clues to understand the pathogenesis of PKCβII-related disorders and facilitate their treatment.