Paxillin在T細胞活化上角色之探討

Abstract

Paxillin是integrin下游重要的轉接蛋白(adaptor protein)之一。Integrin 會幫助T細胞和抗原呈獻細胞的結合，且在T細胞活化時提供共同活化訊息(co-stimulatory signal)。已知paxillin會在T細胞活化過程中被磷酸化，且聚集在immunological synapse的位置。然而paxillin如何參與T細胞的活化，並沒有明確的結論。在本論文中，我們建立表現paxillin磷酸化位置突變蛋白的DO11.10細胞株，探討paxillin對T細胞活化的影響。也建立表現paxillin磷酸化位置突變蛋白的基因轉殖小鼠，於正常T細胞中分析paxillin在T細胞發育及活化所扮演的角色。 研究中發現，表現paxillin的FAK、JNK或p38MAPK單一磷酸化位置突變蛋白的DO11.10 T細胞株，活化後IL-2的產生或與B細胞結合的能力，皆和YFP對照細胞相當。在表現不同組合的paxillin雙磷酸化位置突變蛋白的DO11.10 T細胞株，TCR活化後分泌IL-2的量減少。其中尤其以表現JNK/p38MAPK雙磷酸化位置突變(S178A/S85A)的paxillin影響IL-2產生最顯著。但paxillin 的雙磷酸化位置突變不影響T細胞與B細胞結合能力。 爲探討paxillin在正常T細胞發育及活化所扮演的角色，我們更進一步建立表現paxillin雙磷酸化位置突變(S178A/S85A)蛋白的基因轉殖小鼠。與同胎對照小鼠相比，發現基因轉殖小鼠的T細胞發育有缺陷,胸腺single positive T細胞及脾臟CD4 T細胞族群均降低。另外，S178A/S85A-paxillin基因轉殖小鼠的胸腺或脾臟T細胞，TCR刺激後細胞增生及IL-2分泌，都比同胎對照小鼠減少。 綜合我們的結果，paxillin在p38MAPK、JNK或FAK磷酸化位置的突變組合，明顯抑制T細胞株的活化。阻斷paxillin被p38MAPK及JNK同時磷酸化也抑制正常T細胞的發育和活化。我們的結果顯示paxillin被不同激酶磷酸化，尤其是p38MAPK及JNK，是T細胞活化及發育所必需。

Paxillin is one of the important adaptor proteins down stream of integrin. Integrin supports the association of T cell and antigen presenting cell, and provides co-stimulatory signal during T cell activation. It is known that during T cell activation, paxillin is phosphorylated and recruited to the site of immunological synapse. However, the exact role of paxillin in T cell activation has not been defined. In this study, we elucidated the role of paxillin in T cell activation, by expressing different paxillin phosphorylation site mutant in DO11.10 hybridoma. We also examined the role of paxillin in normal T cell activation and development, by expressing S178A/S85A paxillin double phosphorylation site mutant in transgenic mice. Results from our study demonstrate that overexpression of paxillin with mutation at single phosphorylation site targeted by FAK, JNK, or p38 in DO11.10 hybridoma did not affect T cell activation, as measured by IL-2 production or T cell-antigen presenting cell conjugation. Expression of paxillin with mutation at double phosphorylation site in different combination, resulted in decreased IL-2 production upon TCR stimulation. Expression of paxillin with mutation at double phosphorylation site targeted by JNK and p38 (S178A/S85A), led to the most significant reduction of IL-2 generation. However, T cell-antigen presenting cell conjugation was not altered by expression of paxillin with mutation at double phosphorylation sites. To study the role of paxillin in T cell activation and development in normal T cells, we further generated paxillin S178A/S85A transgenic mice. In comparison with normal littermate control mice, transgenic mice showed defects in T cell development, with reduction in thymic single positive T cell population and in splenic CD4 T cell population. Thymocytes and splenic T cells from S178A/S85A-paxillin transgenic mice, displayed attenuated cell proliferation as well as IL-2 production during TCR stimulation. In summary, mutation of paxillin at phosphorylation sites targeted by combination of p38MAPK, JNK, or FAK clearly inhibited activation of T cell line. In addition, blockage of paxillin phosphorylation by p38MAPK and JNK suppressed activation and development of normal T cells. Our results indicate that phosphorylation of paxillin by different kinases, especially p38MAPK and JNK, is essential for T cell activation and T cell development.