【求助】急急求助答案——关于SB203580抑制P38MAPK的自身磷酸化
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如题,我的实验是缺氧复氧处理脐静脉内皮细胞,一组实验细胞给予适当剂量的P38抑制剂SB203580,在进行缺氧复氧,另外一组不加直接缺氧复氧处理,(另外组此处略去),我得出的结论是加入SB203580预处理的组,其磷酸化的P38的水平显著低于未加SB203580组,也就是它可以抑制P38的磷酸化,我看国内外大部分的实验也是得出的中国结论,但是审稿人却找出了一篇99年BBRC的文献,来质疑我的观点,说The SB203580 inhibitor blocks the activity of p38, i.e. its ability to phosphorylate its substrates. It does not block the phosphorylation of p38 itself. (see kumar et al, 1999, Biochem Biophys Res Commun 268:825-831).
意思是说它不能抑制P38的磷酸化,而是抑制P38对其底物的活化,原始文献我也没怎么看明白,要求一周之内恢复审稿人,我好崩溃啊,请有经验的前辈、高手解答!!!
万分感谢!!!
补充提问,关于P38的自身磷酸化和上游激活其磷酸化,有什么却别,分别见于什么样的情况,用缺氧复氧处理,可以使内皮细胞的P38发生自身磷酸化吗?
谢谢!
谢谢!
审稿人说的没错,SB203580是p38的抑制剂,是抑制p38的激酶活性的,而不是抑制它本身的活化的。
也有文献说可以抑制其自身的磷酸化的
请上传该文献。
kern6549 wrote:
请上传该文献。
不知道我对这篇文献的理解是否正确,PNAS貌似比较牛,但我英语水平有限,麻烦高手看看!
谢谢!
谢谢!
附上审稿人的原始文献。
看了你PNAS的那篇文章,也是说SB203580是抑制p38的活性的呀,什么地方说它能抑制p38自身的磷酸化的,请你指出来,可能是我漏掉了。
给你找了个非常明确说SB203580是抑制p38的活性的证据:
文章第2页图2的文章说明部分的第一句话:Pharmacological inhibition of p38 MAPK activity prevents AL-LC–induced oxidant stress and contractile dysfunction in isolated adult cardiomyocytes.
给你找了个非常明确说SB203580是抑制p38的活性的证据:
文章第2页图2的文章说明部分的第一句话:Pharmacological inhibition of p38 MAPK activity prevents AL-LC–induced oxidant stress and contractile dysfunction in isolated adult cardiomyocytes.
Treatment of cardiomyocytes with SB203580, a selective p38
MAPK inhibitor, significantly attenuated AL-LC–induced oxidative
stress, cellular dysfunction, and apoptosis. Our data provide a
unique mechanistic insight into the pathogenesis of AL-LC cardiac
toxicity and suggest that TAB1-mediated p38α MAPK autophosphorylation
may serve as an important event leading to cardiac
dysfunction and subsequent heart failure.
SB203580 exerts its inhibitory effect by binding to the ATP binding pocket of
p38 MAPK, thus inhibiting its ability to undergo autophosphorylation,
but not affecting the capacity of p38 MAPK to be phosphorylated
by upstream MAPKK, MKK3/6. Taking advantage of
this chemical property, SB203580 has been used to identify the p38
MAPK autophosphorylation in cultured cells in several prior
published studies (13–15, 17, 24).
(可否理解为如果给予SB203580预处理,能够降低P-P38的表达,而上游的MKK3/6没有活化,则说明此处的P38是通过自身磷酸化,而且SB203580可以抑制它的自身磷酸化)
不知道我的理解是否正确,请高手指点,谢谢!
MAPK inhibitor, significantly attenuated AL-LC–induced oxidative
stress, cellular dysfunction, and apoptosis. Our data provide a
unique mechanistic insight into the pathogenesis of AL-LC cardiac
toxicity and suggest that TAB1-mediated p38α MAPK autophosphorylation
may serve as an important event leading to cardiac
dysfunction and subsequent heart failure.
SB203580 exerts its inhibitory effect by binding to the ATP binding pocket of
p38 MAPK, thus inhibiting its ability to undergo autophosphorylation,
but not affecting the capacity of p38 MAPK to be phosphorylated
by upstream MAPKK, MKK3/6. Taking advantage of
this chemical property, SB203580 has been used to identify the p38
MAPK autophosphorylation in cultured cells in several prior
published studies (13–15, 17, 24).
(可否理解为如果给予SB203580预处理,能够降低P-P38的表达,而上游的MKK3/6没有活化,则说明此处的P38是通过自身磷酸化,而且SB203580可以抑制它的自身磷酸化)
不知道我的理解是否正确,请高手指点,谢谢!
可以理解为SB203580是抑制P38的激酶活性的,而它的激酶活性的底物有两种,一种是自己(反映为自身的磷酸化),一种是下游的底物,SB203580能够同时抑制这个过程。
但是SB203580不能影响P38上游的激酶对其的活化过程。
可以说上游的激酶活化P38之后,活化的P38就能够自我活化,就相当于一个正反馈调节,SB203580不能抑制启动过程,但能够抑制这种正反馈调节的过程。
所以,如果这个启动过程没有,P38没有被上游的分子活化,SB203580对P38的活化是没有作用的。
但是SB203580不能影响P38上游的激酶对其的活化过程。
可以说上游的激酶活化P38之后,活化的P38就能够自我活化,就相当于一个正反馈调节,SB203580不能抑制启动过程,但能够抑制这种正反馈调节的过程。
所以,如果这个启动过程没有,P38没有被上游的分子活化,SB203580对P38的活化是没有作用的。
好晕啊 完全没懂:(
我只关心一个结论是否成立
就是如果SB203580能够降低P-P38的表达,是否能说明此处的P38是通过自身磷酸化而活化的
也就是还是存在加入SB203580之后 可以降低P-P38的表达的情况的 比如自身的磷酸化
对吗?
审稿人纠结我为什么加入SB203580之后 怎么会降低P-P38的表达 但是我做出来的结果就是如此
我只关心一个结论是否成立
就是如果SB203580能够降低P-P38的表达,是否能说明此处的P38是通过自身磷酸化而活化的
也就是还是存在加入SB203580之后 可以降低P-P38的表达的情况的 比如自身的磷酸化
对吗?
审稿人纠结我为什么加入SB203580之后 怎么会降低P-P38的表达 但是我做出来的结果就是如此
你可以将第二段的英文引用反映于审稿人,并且审明你的结果确实如此的,而且也与文献中的相符。
另外,SB203580能够降低P-P38的表达,不能说明此处的P38是通过自身磷酸化而活化的。应该说此处的P38是通过两种方式活化的,一个是上游的激酶活化它,一个是自身磷酸化,而SB203580抑制的就是这个自身磷酸化。你要明白,没有上游的激酶活化它启动这个级联反应,它是不会自身磷酸化的,所以有自身磷酸化存在的情况下,肯定有上游的激酶活化它。
另外,SB203580能够降低P-P38的表达,不能说明此处的P38是通过自身磷酸化而活化的。应该说此处的P38是通过两种方式活化的,一个是上游的激酶活化它,一个是自身磷酸化,而SB203580抑制的就是这个自身磷酸化。你要明白,没有上游的激酶活化它启动这个级联反应,它是不会自身磷酸化的,所以有自身磷酸化存在的情况下,肯定有上游的激酶活化它。
好的 谢谢
p38阻断剂有SB203580和SB202190两种,是否后者可以直接抑制P38的磷酸化?对SB203580的描述:A highly specific, cell-permeable inhibitor of p38 kinase (IC50 = 34 nM in vitro, 600 nM in cells). Also known as reactivating kinase (RK) and CSBP (cytokine synthesis anti-inflammatory drug binding protein). Does not significantly inhibit JNK or p42 MAP kinase even at 100 µM. Inhibits IL-1 and TNF-α production from LPS-stimulated human monocytes and the human monocyte cell line THP-1 (IC50 = 50-100 nM). SB 203580 has also been shown to be an effective inhibitor of inflammatory cytokine production in vivo in both mice and rats.
对SB202190的描述:A potent and cell-permeable inhibitor of p38 MAP kinase. Inhibits p38 phosphorylation of myelin basic protein (MBP) with no effect on the activity of the ERK or JNK MAP kinase subgroups. Also inhibits the kinase activity of p38β (Ki = 16 nM; IC50 = 350 nM) and p38 phosphorylation of activating transcription factor 2 (ATF-2; IC50 = 280 nM). Blocks LPS-induced TNF-α and interleukin biosynthesis. Reported to induce LDL receptor expression in Hep52 cells.
对SB202190的描述:A potent and cell-permeable inhibitor of p38 MAP kinase. Inhibits p38 phosphorylation of myelin basic protein (MBP) with no effect on the activity of the ERK or JNK MAP kinase subgroups. Also inhibits the kinase activity of p38β (Ki = 16 nM; IC50 = 350 nM) and p38 phosphorylation of activating transcription factor 2 (ATF-2; IC50 = 280 nM). Blocks LPS-induced TNF-α and interleukin biosynthesis. Reported to induce LDL receptor expression in Hep52 cells.
从对SB202190的描述来看,没有说明SB202190可以直接抑制P38的磷酸化。只是说它能够抑制P38对MBP和ATF-2的磷酸化。
哦 那这两种应该是差不多的类似物了
谢谢楼上的 呵呵 MAPK高手
谢谢楼上的 呵呵 MAPK高手
一般用p38的抑制剂,检测的比较多的是下游kinase的磷酸化情况,或者其他相关蛋白的情况,比如SRP72 protein。直接用SB-203580 来研究p38的磷酸化情况,不很适宜。
你自己提供的那篇文章将的非常清楚,“Pyridinylimidazole Compound SB 203580 Inhibits the Activity but Not the Activation of p38 Mitogen-Activated Protein Kinase”,在abstract中说的是,SB-203580是不是通过结合到p38的磷酸化位点来抑制p38活性的,而是抑制p38的底物MAPKAP K2的活性,来抑制这个反应的。原文“SB 203580,however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK.”
我又check了那篇pnas的文章,作者其实有2段话来描述影响情况。这一段话是楼主忽略的:
“the enhancement of p38 MAPK phosphorylation by AL-LC treatment was significantly attenuated by the presence of SB203580 (5 μM); however, inhibition of p38 MAPK activity by SB203580 did not change the basal level of p38 phosphorylation, suggesting that constitutive activation of MKK3/6 may contribute to the basal level of p38 MAPK activation that is independent of AL-LC–triggered p38 MAPK phosphorylation.”
在加上楼主的那一段:
“Using immunoprecipitation of TAB1 with TAB1-specific antibody from total cell lysate followed by
immunoblotting with anti-TAB1 and anti-p38 antibodies, we found an increase in endogenous TAB1 and p38 MAPK interaction in cells treated with AL-LC in comparison with Con-LC and vehicle-treated groups (Fig. 3D). Taken together, these results suggest that TAB1-mediated p38 autophosphorylation rather than canonical p38 MAPK activation is an early event in AL-LC–induced cellular stress and dysfunction.”
在这之前,文章首先强调了,“Emerging data suggest that TAB1 may recruit activated p38, which can result in p38 autophosphorylation.”p38的自我磷酸化是由于TAB-1引起的,不知道楼主有没有在自己的实验中,证明你自己的细胞株中TAB-1的情况,如果没有,p38不会自动的发生自身磷酸化。
另外,这2段话,强调了,SB203580在抑制p38活性的时候,是抑制掉了由于AL-LC treatment 引起的p-p38的增加量,但是,本来是不改变p-p38的基础水平。这是由于p38的自身磷酸化,是不受SB203580影响的。
这恰恰证明了,SB203580不能影响p38的自身磷酸化。这个结果也正好与BBRC这篇文章相符合,正是因为SB203580只能影响p38的底物,所以,SB203580不能对p38的自身磷酸化产生作用。
不知道这样的回答能不能解决你的疑问,呵呵。
我是selleckchem中国区域的技术支持,SB203580也正好是公司的产品。
你自己提供的那篇文章将的非常清楚,“Pyridinylimidazole Compound SB 203580 Inhibits the Activity but Not the Activation of p38 Mitogen-Activated Protein Kinase”,在abstract中说的是,SB-203580是不是通过结合到p38的磷酸化位点来抑制p38活性的,而是抑制p38的底物MAPKAP K2的活性,来抑制这个反应的。原文“SB 203580,however, potently inhibited the activity of p38 MAPK as demonstrated by the inhibition of the activation of MAPKAP K2, a specific physiological substrate of p38 MAPK.”
我又check了那篇pnas的文章,作者其实有2段话来描述影响情况。这一段话是楼主忽略的:
“the enhancement of p38 MAPK phosphorylation by AL-LC treatment was significantly attenuated by the presence of SB203580 (5 μM); however, inhibition of p38 MAPK activity by SB203580 did not change the basal level of p38 phosphorylation, suggesting that constitutive activation of MKK3/6 may contribute to the basal level of p38 MAPK activation that is independent of AL-LC–triggered p38 MAPK phosphorylation.”
在加上楼主的那一段:
“Using immunoprecipitation of TAB1 with TAB1-specific antibody from total cell lysate followed by
immunoblotting with anti-TAB1 and anti-p38 antibodies, we found an increase in endogenous TAB1 and p38 MAPK interaction in cells treated with AL-LC in comparison with Con-LC and vehicle-treated groups (Fig. 3D). Taken together, these results suggest that TAB1-mediated p38 autophosphorylation rather than canonical p38 MAPK activation is an early event in AL-LC–induced cellular stress and dysfunction.”
在这之前,文章首先强调了,“Emerging data suggest that TAB1 may recruit activated p38, which can result in p38 autophosphorylation.”p38的自我磷酸化是由于TAB-1引起的,不知道楼主有没有在自己的实验中,证明你自己的细胞株中TAB-1的情况,如果没有,p38不会自动的发生自身磷酸化。
另外,这2段话,强调了,SB203580在抑制p38活性的时候,是抑制掉了由于AL-LC treatment 引起的p-p38的增加量,但是,本来是不改变p-p38的基础水平。这是由于p38的自身磷酸化,是不受SB203580影响的。
这恰恰证明了,SB203580不能影响p38的自身磷酸化。这个结果也正好与BBRC这篇文章相符合,正是因为SB203580只能影响p38的底物,所以,SB203580不能对p38的自身磷酸化产生作用。
不知道这样的回答能不能解决你的疑问,呵呵。
我是selleckchem中国区域的技术支持,SB203580也正好是公司的产品。
受用啊
P38MAPK信号通路阻断剂种类繁多,最常见的是一种啦略咪啤芳基杂环类复合物,常用的有SB203580、SB202190、SB220025等,它们在在p38 MAPK信号通路的研究中的有效性均已经被充分证明。其阻断机理不是作用于其上游激酶或下游作用底物,而是通过直接或间接竞争性与p38a、P亚基上的具有ATP酶活性的结合位点结合,使P38MAPK失去与ATP结合的能力,从而使其失去激酶活性,实现对P38MAPK信号通路的抑制作用[1]。
[1] Lisnock J,Tebben A,Frantz B,et al. Molecular basis for p38 protein kinase inhibitor
specificity [J] .Biochemistry, 1998,3(47): 16573-16581.
请问:其阻断机理不是作用于其上游激酶或下游作用底物又如何理解呢?
[1] Lisnock J,Tebben A,Frantz B,et al. Molecular basis for p38 protein kinase inhibitor
specificity [J] .Biochemistry, 1998,3(47): 16573-16581.
请问:其阻断机理不是作用于其上游激酶或下游作用底物又如何理解呢?
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