JNK 及 p38 MAPK 磷酸化 paxillin 在 T細胞活化上之角色研究

Abstract

Paxillin 是大小為68-kD的細胞骨架轉接蛋白，可連結至focal adhesion複合體。它具有許多可和其他蛋白交互作用的區塊，並藉此整合外來訊息並加以傳遞。近年來有許多研究指出，paxillin 蛋白上絲胺酸及酪胺酸的磷酸化情形對其功能相當重要，但paxillin的磷酸化在T細胞的活化上所扮演的角色目前並不清楚。在本研究中，我們建立表現 paxillin 上FAK、JNK及p38 MAPK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。 在我們的實驗中發現，雖然paxillin 在上皮細胞及神經元細胞的延展及遷徙扮演關鍵角色，在 DO11.10 細胞中表現 paxillin 的突變株並不影響細胞的integrin黏附到其基質的能力，且只有表現FAK 磷酸化位置 paxillin 突變株細胞之遷徙能力受到些微的抑制。雖然JNK 和 p38 MAPK 磷酸化 paxillin 並不影響T細胞的移動，在表現 JNK/p38MAPK 雙磷酸化位置paxillin 突變株的 DO11.10 細胞中，發現TCR 活化後 IL-2 的產量明顯減少，而這個現象可歸因於轉錄因子NFAT的進核受到抑制。 為了更深入探討paxillin 對於正常T細胞的活化有何影響，我們也建立表現 paxillin磷酸化位置突變蛋白的基因轉殖小鼠。和同胎對照小鼠相比，基因轉殖小鼠的T細胞的發育並無明顯差異，細胞的黏附和遷徙也不受影響，但活化後細胞增生的速度及產生IL-2的能力都受到抑制。在基因轉殖小鼠中同樣可觀察到NFAT 進入細胞核的量明顯減少，除了影響IL-2 的分泌，IFN-gamma 及 IL-4 的產生也受到影響。 綜合以上實驗結果，同時在paxillin的p38MAPK及JNK 磷酸化位置進行突變，可明顯抑制T細胞的活化。而被p38 MAPK 及 JNK所活化的paxillin 是如何調控NFAT 的進核以及T細胞的活化，則有待進一步的實驗探討。

Paxillin is a 68-kD cytoskeletal adaptor protein associated with focal adhesion complex. It is a multidomain adaptor that facilitates signal integration and transduction. Recent studies revealed critical roles for tyrosine and serine phosphorylation of paxillin by FAK, JNK and p38 MAPK, but the consequence of paxillin phosphorylation in T cells remains unknown. In this study, we addressed this issue by overexpressing paxillin mutants with respective phosphorylation sites of FAK, JNK, and p38 MAPK in T cells, and examined the role of paxillin phosphorylation in T cell activation, adhesion, and migration. Contradictory to the reported effect on epithelial cells and neurite, all the paxillin mutants examined did not interfere with integrin-mediated T-cell adhesion. Overexpression of paxillin with mutations at phosphorylation sites of FAK (Y31F/Y118F) in T cells reduced SDF-1alpha--stimulated migration, but did not affect T cell activation, and other parameters of T cell activation remain normal. In contrast, overexpression of paxillin with double mutations at phosphorylation sites of p38 MAPK and JNK (S85A/S178A) in T cells did not alter cell adhesion and migration, but effectively suppressed IL-2 production, the signature of T cell activation. Inhibition by [S85A/S178A]-paxillin could be partly attributed to a selective suppression of NFAT activation. To study the role of paxillin in T cell activation in normal T cells, we further generated [S85A/S178A]-paxillin transgenic mice. Although no significant effect of [S85A/S178A]-paxillin on T cell development was observed, both T cell proliferation and IL-2 production were suppressed in transgenic mice compared to NLC mice. In addition, NFAT translocation was partially interfered by [S85A/S178A]-paxillin transgene. In summary, phosphorylations of paxillin by different kinases play different roles in T cells and non-T cells. How [S85A/S178A]-paxillin modulates T cell activation and NFAT activation will be further investigated.