Profiling the Dynamics of a Human Phosphorylome Reveals New Components in HGF/c-Met Signaling

Protein phosphorylation is a dynamic and reversible event that greatly influences cellular function. Identifying the key regulatory elements that determine cellular phenotypes during development and oncogenesis requires the ability to dynamically monitor proteome-wide events. Here, we report the development of a new strategy to monitor dynamic changes of protein phosphorylation in cells and tissues using functional protein microarrays as the readout. To demonstrate this technology's ability to identify condition-dependent phosphorylation events, human protein microarrays were incubated with lysates from cells or tissues under activation or inhibition of c-Met, a receptor tyrosine kinase involved in tissue morphogenesis and malignancy. By comparing the differences between the protein phosphorylation profiles obtained using the protein microarrays, we were able to recover many of the proteins that are known to be specifically activated (i.e., phosphorylated) upon c-Met activation by the hepatocyte growth factor (HGF). Most importantly, we discovered many proteins that were differentially phosphorylated by lysates from cells or tissues when the c-Met pathway was active. Using phosphorylation-specific antibodies, we were able to validate several candidate proteins as new downstream components of the c-Met signaling pathway in cells. We envision that this new approach, like its DNA microarray counterpart, can be further extended toward profiling dynamics of global protein phosphorylation under many different physiological conditions both in cellulo and in vivo in a high-throughput and cost-effective fashion.蛋白质磷酸化是一类动态可逆的生物学事件，对细胞功能具有显著调控作用。要鉴定发育与肿瘤发生过程中决定细胞表型的关键调控元件，需具备动态监测全蛋白质组（proteome）范围内事件的能力。本研究开发了一种以功能蛋白质微阵列（functional protein microarray）为检测读出平台的全新策略，用于监测细胞与组织内蛋白质磷酸化的动态变化。 为验证该技术识别条件依赖性磷酸化事件的能力，我们将人源蛋白质微阵列与经c-Met——一种参与组织形态发生与恶性转化的受体酪氨酸激酶——激活或抑制状态下的细胞或组织裂解液共孵育。通过比对蛋白质微阵列检测得到的蛋白质磷酸化谱间差异，我们成功筛选出诸多已知可在肝细胞生长因子（hepatocyte growth factor, HGF）介导的c-Met激活过程中被特异性激活（即磷酸化）的蛋白质。尤为关键的是，我们发现了诸多在c-Met通路激活时，可被不同细胞或组织裂解液诱导产生差异化磷酸化修饰的蛋白质。利用磷酸化特异性抗体，我们验证了数种候选蛋白质可作为c-Met信号通路新的下游效应分子存在于细胞中。 我们展望，该新方法可与DNA微阵列技术一样得到进一步拓展，以高通量、低成本的方式，在多种不同生理条件下对细胞内与活体体内的全蛋白质组磷酸化动态谱进行系统性分析。