Jang Se Hwan, Jun Chang-Duk, Park Zee-Yong
School of Life Sciences, Gwangju Institute of Science & Technology, 123, Cheomdangwagi-Ro, Buk-Gu, 500-712 Gwangju Republic of Korea.
Proteome Sci. 2015 Mar 26;13:14. doi: 10.1186/s12953-015-0070-9. eCollection 2015.
Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms.
Here, a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and label-free relative quantification analysis was used to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signalling pathways of immune cell activation and homeostasis. A total of six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.
Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes. Accordingly, different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signalling events under immune response activation or homeostasis conditions.
转胶蛋白2是一种细胞骨架肌动蛋白结合蛋白，最近有研究表明其参与免疫突触的形成。尽管转胶蛋白2的详细功能在很大程度上尚不清楚，但转胶蛋白2与肌动蛋白之间的相互作用似乎在调节转胶蛋白2的细胞功能中起着重要作用。由于蛋白质磷酸化可以改变与其他蛋白质相互作用的能力，因此对转胶蛋白2进行全面的磷酸化分析将有助于理解其功能机制。
在此，我们使用了一种结合免疫沉淀、IMAC磷酸肽富集技术和无标记相对定量分析的特异性蛋白质无标记定量磷酸化分析方法，来监测在蛋白激酶C（PKC）和蛋白激酶A（PKA）激活条件下，Jurkat T细胞中过表达的转胶蛋白2的磷酸化变化，这两种条件分别代表免疫细胞激活和稳态的两种典型细胞内信号通路。总共鉴定出六个丝氨酸/苏氨酸磷酸化位点，包括一个新的磷酸化位点苏氨酸-84。值得注意的是，在两种激酶激活条件下观察到了转胶蛋白2不同的磷酸化模式。大多数显示出特定激酶依赖性磷酸化变化的磷酸化位点分别位于两个先前已鉴定的肌动蛋白结合区域：肌动蛋白结合位点（ABS）和钙调蛋白重复结构域（CNR）。PKC激活增加了CNR中苏氨酸-180和丝氨酸-185的磷酸化，而PKA激活增加了ABS中丝氨酸-163的磷酸化。
转胶蛋白2的多个肌动蛋白结合区域共同发挥作用以实现其完整的肌动蛋白结合能力，并且每个肌动蛋白结合区域的肌动蛋白结合亲和力似乎受到特定激酶依赖性磷酸化变化的调节。因此，在免疫反应激活或稳态条件下，不同的细胞内信号事件可能导致转胶蛋白2具有不同的肌动蛋白结合特性或细胞功能。
