Fritz Anne, Brayer Kathryn J, McCormick Nathaniel, Adams Deanna G, Wadzinski Brian E, Vaillancourt Richard R
Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tucson, Arizona 85721, USA.
J Biol Chem. 2006 Mar 10;281(10):6236-45. doi: 10.1074/jbc.M509249200. Epub 2006 Jan 9.
MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.
丝裂原活化蛋白激酶激酶激酶3（MEKK3）是一种丝裂原活化蛋白激酶激酶激酶（MAP3K），在丝裂原活化蛋白激酶和IκB激酶的上游发挥作用。磷酸化被认为是MEKK3依赖性信号转导的关键组成部分，但关于这种MAP3K的磷酸化位点知之甚少。为了解决这个问题，在激活环（T环）中引入点突变，用丙氨酸取代丝氨酸或苏氨酸，然后将突变体转染到HEK293爱泼斯坦 - 巴尔病毒核抗原细胞中。MEKK3依赖性激活NF-κB报告基因以及ERK、JNK和p38丝裂原活化蛋白激酶与526位丝氨酸的需求相关。由S526D和S526E组成的MEKK3组成型活性突变体能够激活NF-κB荧光素酶报告基因以及ERK和MEK，这表明Ser526处的负电荷是MEKK3活性所必需的，并暗示Ser526是一个磷酸化位点。开发了一种抗体，它能特异性识别MEKK3的磷酸化Ser526，但不能识别S526A点突变体。MEKK3的催化失活（K391M）突变体在Ser526处未被磷酸化，这表明Ser526的磷酸化是通过自磷酸化发生的。内源性MEKK3在渗透压应激下在Ser526处被磷酸化。此外，体外MKK6磷酸化需要Ser526的磷酸化，而Ser526的去磷酸化由蛋白磷酸酶2A介导，并且对冈田酸和氟化钠敏感。最后，MEKK3与14-3-3的结合依赖于Ser526，并阻止了Ser526的去磷酸化。总之，MEKK3的Ser526是T环内的一个自磷酸化位点，受PP2A和14-3-3蛋白调控。
