Institute of Pathology, University of Bern , Bern , Switzerland.
PeerJ. 2013 Aug 29;1:e131. doi: 10.7717/peerj.131. eCollection 2013.
We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90(-/-) mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.
我们之前已经确定了磷脂酰肌醇-4-磷酸 5-激酶 I 型（PIPKI）γ90 是 T 细胞尾足的组成部分。然而，其定位的分子决定因素和功能后果仍然未知。在本报告中，我们试图更好地理解 PIPKIγ90 尾足靶向的机制以及 PIPKIγ90 在 T 细胞尾足形成中的作用。在 T 细胞激活过程中，PIPKIγ90 与膜微区相关蛋白 flotillin-1 和 -2 共定位，并积累在尾足中。我们报告说，PIPKIγ90 的 C 端 26 个氨基酸延伸对于其在尾足中的定位是必需的。我们进一步使用 PIPKIγ90(-/-) 小鼠的 T 细胞和表达激酶失活 PIPKIγ90 突变体的人 T 细胞来研究 PIPKIγ90 在 T 细胞尾足形成中的作用。我们发现 PIPKIγ90 缺陷型 T 细胞在 ICAM-1 上具有伸长的尾足。此外，在人 T 细胞中过表达自然存在的缺少最后 26 个氨基酸的同工型 PIPKIγ87，会抑制尾足的形成并损害尾足蛋白如 flotillin 的加帽。转染人 T 细胞中的 flotillin-2 显性失活突变体，转而减弱 PIPKIγ90 的加帽。我们的数据有助于理解调节 T 细胞尾足形成的分子机制。
