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주일로(Ilo Jou),고성수(Sung Soo Ko),안영수(Young Soo Ahn) 대한약리학회 1993 대한약리학잡지 Vol.29 No.1
흰쥐 말초 T림프구에 분열유발 물질인 PMA와 Con A를 투여하여 인산화되는 단백을 확인하고, PKC 억제제인 H-7, CaM kinase 억제제인 W-7을 전처치한 후의 인산화 변동과 시간 경과에 따른 인산화 변동을 관찰하였다. 그 결과 흰쥐 T림프구를 PMA로 자극하면 5개의 인산화 단백이 새로이 나타나고 7개 단백의 인산화가 증가 되었으며, Con A자극으로는 1개의 단백이 새로이 인산화 되고 7개 단백의 인산화가 증가되었다. PMA 및 Con A자극으로 인산화 되는 13개 단백은 kinase억제제 전처치에 의하여 3군으로 각각 구분되며, H-7 전처치로 24 kDa/pI 7.1, 24/7.2, 26/6.1, 74/6.2 단백의, W-7 전처치로 14 kDa/pI5.9, 28/6.8, 29/6.9, 28/7.0, 44/6.8, 58/6.2 단백의 인산화가 현저히 감소 되었으며, 18 kDa/p1 5.4, 25/7.3 및 54/5.2단백은 두 억제제에 의해 영향을 받지 않았다. 이들 인산화 단백은 대부분 세포의 soluble fraction에서 확인되며 자극후 반응 초기에 인산화 된 후 인산화가 감소하나, 침전물에서 관찰되는 소수의 인산화 단백은 지속적인 인산화를 보였다. 한편 Kinase 억제제 처리에 의하여 구분된 3군에 속하는 단백들의 시간에 따른 인산화 양상을 관찰한 결과 각 군에 따른 인산화 양상에 상호 연관성이 없었다. 이상의 실험결과로 보아 림프구 활성의 초기 단계에서 인산화 되는 단백에는 PKC, CaM kinase 및 다른 kinase에 의해 인산화 되는 3종류의 단백이 존재하며, 3종류의 kinase의 활성은 단계적인 활성이 아니라 독립적 또는 상호 협동적으로 작용하여 림프구 활성을 유발시키는 것으로 생각된다. This study was done to classify the proteins involved in the specific phosphorylation using the rat peripheral blood lymphocytes (rPBL) stimulated with mitogens, phorbol 12-myristate 13-acetate (PMA) and concanavalin A (Con A). The lymphocytes were incubated with <sup>32</sup>P-orthophosphate before PMA or Con A stimulation. The migration patterns of the phosphorylated proteins of mitogen-treated rPBL in two dimensional electrophoretic fields were analyzed after autoradiography. The stimulation of the lymphocytes with PMA and Con A increased the phosphorylation of thirteen protein fractions. The phosphorylation intensities of the protein spots differ to the treatments of the cells with specific kinase inhibitors, H-7 and W-7. These protein fractions were grouped into 3 classes, namely, PKC-mediated, CaM kinase-mediated, and other kinase mediated proteins. The effect of the duration of the stimulation on the phosphorylated behaviors occurred concurrently, not sequentially, although each individual protein fraction had a different time for the peak phosphorylation during the stimulation period upto 30 minutes. The phosphoproteins found in the cytosolic soluble fraction were phosphorylated prior to those in the pellet, whose phosphorylations were sustained at a high level for over 10 minutes. The above results suggest that the early events in lymphocyte activation involve 3 different sets of proteins which are phosphorylated by CaM kinase, PKC and other kinases, and those kinases do not work sequentially, but rather, independently or cooperatively.
흰쥐 말초 림프구의 분자량 22 kDa 인산화 단백질의 특성
주일로,권혁춘,안영수 아주대학교 의과학연구소 1996 아주의학 Vol.1 No.1
Lymphocytes were found to possess all of the enzymatic machinery needed to phosphorylate and dephosphorylate proteins. At least four groups of protein kinases participate in T cell activation and interact with each other in a complex and as yet unknown manner. Increased phosphorylation in lymphocytes following stimulation with mitogens or interleukin-2, and the pivotal role of protein kinase C(PKC) in the initial biochemical reaction have been reported. But the exact role of PKC in T cell activation and the substrate of PKC are not well known. This study was attempted to clarify the characteristics of 22 KDa phosphoprotein obtained from rat peripheral blood lymphocytes(rPBL) stimulated with phorbol 12-myristale 13-acetate(PMA), using various kinase inhibitors as well as kinase activators. The lymphocytes were incubated with ^(32)P orthophosphate before PMA stimulation. The migration pattern of the phosphorylated proteins of PMA-treated rPBL in the two dimensional electrophortic fields were analyzed after autoradiography. And the phosphorylation sites of 22 kDa protein were analyzed by high performance liquid chromatography(HPLC) and scintilation counting. The results are as follows: 1) Increased phosphorylation of 22 kDa protein was observed with PMA. 2) Forskolin, an activator of adenylyl cyclase, causes no significant change of phosphorylation of 22 kDa. 3) A 23187, a Ca^(2+) ionophore, has no noticed effect on the phosphorylation of 22 kDa protein. 4) Staurosporine, a potent PKC inhibitor, showed inhibitory action on PMA-stimulated phosphorylation of 22 kDa. 5) Calphostin C, specific PKC inhibitor inhibited the PMA-stimulated phosphorylation of 22 kDa. 6) Among trypic peptide fractions of 22 kDa by HPLC, one ^(32)P phosphopeptide peak was observed at about 20% acetonitrile. From the above results, it could be suggested that the 22 kDa phosphoprotein of rat peripheral blood lymphocytes would be a substrate of PKC, and has one phosphorylation on serine residue.
최인섭,조은혜,변지원,박상면,Ilo Jou 한국뇌신경과학회 2016 Experimental Neurobiology Vol.25 No.5
Mutation of leucine-rich repeat kinase 2 (LRRK2) causes an autosomal dominant and late-onset familial Parkinson’s disease (PD). Recently, we reported that LRRK2 directly binds to and phosphorylates the threonine 474 (T474)-containing Thr-X-Arg(Lys) (TXR) motif of focal adhesion kinase (FAK), thereby inhibiting the phosphorylation of FAK at tyrosine (Y) 397 residue (pY397-FAK), which is a marker of its activation. Mechanistically, however, it remained unclear how T474-FAK phosphorylation suppressed FAK activation. Here, we report that T474-FAK phosphorylation could inhibit FAK activation via at least two different mechanisms. First, T474 phosphorylation appears to induce a conformational change of FAK, enabling its N-terminal FERM domain to autoinhibit Y397 phosphorylation. This is supported by the observation that the levels of pY397-FAK were increased by deletion of the FERM domain and/or mutation of the FERM domain to prevent its interaction with the kinase domain of FAK. Second, pT474- FAK appears to recruit SHP-2, which is a phosphatase responsible for dephosphorylating pY397-FAK. We found that mutation of T474 into glutamate (T474E-FAK) to mimic phosphorylation induced more strong interaction with SHP-2 than WT-FAK, and that pharmacological inhibition of SHP-2 with NSC-87877 rescued the level of pY397 in HEK293T cells. These results collectively show that LRRK2 suppresses FAK activation through diverse mechanisms that include the promotion of autoinhibition and/or the recruitment of phosphatases, such as SHP-2.