Adaptor Protein, Lad, associates with G protein β subunit and mediates Chemokine-dependent migration of T cells

Abstract

Part1 Previously, Lad was identified as an adaptor protein playing essential roles in TCR-mediated T cell activation through association with the SH2 domain of p56^(Lck). Furthermore, in yeast two-hybrid screening with Lad as bait, G protein β subunit (Gβ) was discovered as the binding partner of Lad. As the most prominent G protein coupled receptor in T cells is chemokine receptor, the finding suggested the involvement of Lad involved in chemokine signaling. Subsequent experiments revealed that in response to chemokine treatment, Lad inducibly associated with Gβ and was tyrosine phosphorylated in Jurkat T cells. Furthermore, knockdown of endogenous Lad expression by SiRNA resulted in the impairment of chemokine-induced migration of T cells indicating that Lad is required for the chemokine-induced T cell migration. Here, I studied the Lad-mediated downstream signaling pathways of chemokine receptor and found that; 1) Under confocal microscopy, Lad co-localized with Gβ subunit in response to chemokine. 2) Lad associated with p56^(Lck) and ZAP-70 in response to chemokine treatment, indicating that Lad acts as a platform for the recruitment of two tyrosine kinases to Gβ signaling complex. Furthermore, upon reducing Lad expression by SiRNA expression, chemokine-induced phosphorylation of ZAP-70 was down-regulated. These results show that Lad is required for the chemokine-induced phosphorylation of ZAP-70. It seems that Lad plays a role as a linker between Lck and ZAP-70 and promotes the phosphorylation of ZAP-70 by p56^(Lck). Once activated, ZAP-70 as well as p56^(Lck) may phosphorylate various downstream signaling molecules of chemokine signaling pathway. 3) The chemokine-induced phosphorylation of p42/44 MAPK was completely down-regulated in Lad SiRNA-expressing cells. Moreover, the chemokine-induced activation of p42/44 MAPK was increased in the Jurkat T cell clones expressing Lad but decreased in clones SH2 domain of Lad (a dominant negative form of Lad by its ability impair TCR-mediated IL-2 promoter activation). These data show that Lad is required for chemokine-induced p42/44 MAPK activation. 4) The chemokine-induced phosphorylations of focal adhesion molecules such as Pyk2, paxillin and Crk were downregulated in Lad SiRNA-expressing cells. Therefore, Lad is required for the activation of focal adhesion complexes in response to chemokine. In summary Lad, upon chemokine stimulation, acts as an adaptor protein linking G protein β subunit to tyrosine kinase p56^(Lck) and ZAP-70 and facilitates the phosphorylation of ZAP-70 by p56^(Lck). Thereby, Lad promotes the activation of downstream molecules related to chemokine signaling, including p42/44 MAPK and focal adhesion complexes such as Pyk2, paxillin and Crk. Part2 One of the greatest properties in immune response is that the secondary immune response is stronger and faster upon recountering Ag than the primary immune response and this phenomenon is called of 'immunological memory'. Immunological memory is performed by memory T and B cells which can be long-lived and can induce faster immune response compared to naive cells. To understand the long-living property of CD8^(+) memory T cells, we compared the mRNA levels of various genes involved in cell cycle regulation in naive, effector and memory T cells. Semi-quantitative RT-PCR was performed using total RNA from naive, effector and memory CD8 T cells generated in the mice infected with LCMV (lymphocytic choriomeningtis virus) at various time point. Here, we focused on cell cycle regulator molecules including p53 pathway-related molecules (p53, p21 and MDM2) and CDKs inhibitors such as p16, p19 and p27. We found that p53, p19 and MDM2 were expressed at equal level but the expression levels of p21 and p16 were down-regulated during transition from effector T cells to memory T cells, suggesting that memory T cells have diminished susceptibility to senescence possibly due to downregulation of p21 and p16 levels during effector to memory transition. Recent reports show that Bcl6 is a potent inhibitor of senescence by preventing p19^(ARF)-p53 pathway and Bcl6 plays a critical role in the generation and the maintenance of memory T cells. We found that Bcl6 was co-localized with PML(Promyelocytic Leukemia) and modified with SUMO-1. These data are suggestive that Bcl6 may contribute to memory CD8^(+) T cells formation by overriding senescence pathways mediated by p53 in PML body. In summary, These results suggest that memory CD8^(+) T cells escape the senescence state, in part, by down regulating the senescence-inducing gene expression. ;PART I: Adaptor 단백질인 Lad 는 Gβ와 결합함으로써 chemokine 에의해 유도되는 T 임파구의 이동을 조절한다. 기존의 연구에 의하면 Lad 는 Lck 의 SH2 도메인(domain)과 결합을 통하여 TCR 을 통한 T 임파구의 활성화에 중심적인 역할을 한다고 밝혀져 있었다. Lad 의 기능을 규명하기위하여 Lad 를 bait 로 하여 yeast two hybrid 를수행하였으며, 결합하는 partner 로써 Gβ가 발견되었다. Gβ는 T 임파구에서 가장 잘 알려진 chemokine 의 수용체에 결합하고 있는 effector 단백질이다. 그러므로 Lad 는 T 임파구의 chemokine signaling 에 관여할 것으로 예측되었다. Jurkat T 임파구에 chemokine 을 처리시에 Lad 는 Gβ와 결합하였으며, 동시에 타이로신(tyrosine) 인산화가 되는 것을 확인할 수 있었다. 또한 SiRNA 를 이용하여 T 임파구에 있는 Lad 의 발현을 감소시키면, chemokine 에 반응하여 T 임파구의 이동은 감소하였다. 따라서 Lad 는 chemokine 에 의해 유도되는 T 임파구의 이동을 조절하는 단백질임을 알 수 있었다. 이에 관련된 세포신호전달과정을 알아보기 위해 실험한 결과, Lad 는 tyrosine kinase 인 Lck 와 ZAP-70 와 동시에 결합함으로써 Lck 가 ZAP-70 를 인산화시키는 것을 촉진시키는 동시에 docking 단백질로써 작용한다. 또한 Lad 는 chemokine signaling 의 Downstream molecule 인 MAPkinase 와 focal adhesion complex 의 활성화를 조절하는 것으로 밝혀졌다. PART II : 기억CD8 T 림프구 (CD8 memory T cells)에서 의 세포주기 관련 유전자들의 발현양상 조사 면역학적 기억(Immunological memory )은 동물에서 같은 항원에 재노출 시 빠르고 강한 반응을 의미한다. 또한 기억CD8 T 림프구는 바이러스, 세포 내 감염 세균들의 재침입에 대해 방어하기때문 vaccination의 중요한 목적이 된다. 기억 T 림프구 층은 안정하게 유지되고, 세포생존, 사멸, 증식 간의 평형으로 이러한 항상성은 유지된다. 기억 T 림프구는 원시(naive) T 림프구 의 세포표면 표지인 CD62L^(high) CD44^(low), 작동 (effector) T 림프구의 세포표면 표지인 CD62L^(low) CD44^(high)와 구별되는 세포표면 표지 CD62L (L-selectin) ^(high) CD44^(high)를 발현한다. 우리는 기억 T 림프구의 오래 사는 성질을 이해하기위해, 기억 T 림프구, 원시(naive) T 림프구, 작동 (effector) T 림프구의 서로 다르게 발현하는 분자들을 비교하였다. 특히 세포주기를 조절하는 CDK 저해제에 관심을 가져, semi-quantitative RT-PCR의 방법으로 이러한 유전자의 발현수준 을 측정했다. 실험 7-,40-일전에 LCMV를 감염시킨 쥐(mice)에서 FACS sorter를 이용해 CD62LCD44세포 표면표지를 측정해 원시(Naive), 작동(effector), 기억(memory) CD8 T 림프구를 분리했다. 분리된 세포에서 mRNA를 분리해 semi-quantitative RT-PCR의 주형(templates)으로 사용했다. 이 실험에서 기억(memory) CD8 T 림프구가 작동 (effector) CD8 T림프구보다 훨씬 적은 양의 세포노화를 유도하는 유전자인 p16과 p21를 발현하는 것을 알 수 있었다. 이러한 발견은 기억 (memory) CD8 T 림프구가 오래 살지만, 세포노화를 겪지 않는 다는 것을 보여준다. 또한 Bcl6는 기억(memory) CD8 T 림프구의 생성과 유지에 중요한게 작용한다고 알려져 있다. 최근의 발표에 의하면 Bcl6는 p53-p19 pathway를 억제함으로써 세포노화를 저해한다는 보고가 있다. 우리는 세포노화를 조절하는 단백질 복합체인 PML체에 p53이 들어가 세포노화를 일으키므로, Bcl6가 PML체에 속하는지를 보기위해 293T 세포주를 이용한 결과 Bcl6가 PML과 co-localization되는 것을 확인하였다. 또한 PML체를 구성하는 대부분의 구성 성분들은 sumoylation에 의해 변형이 되므로 Bcl6의 변형여부를 조사한 결과 Bcl6가 sumoylation이 된다는 것을 밝혔다.