The kinase activation loop is the key to mixed lineage kinase-3 activation via both autophosphorylation and hematopoetic progenitor kinase 1 phosphorylation
The kinase activation loop is the key to mixed lineage kinase-3 activation via both autophosphorylation and hematopoetic progenitor kinase 1 phosphorylation
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DOI：
10.1074/jbc.m004092200
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发表时间：
2001-01-19
影响因子：
4.8
通讯作者：
Lassam, N
中科院分区：
文献类型：
--
作者：
Leung, IWL;Lassam, N
我们以前已经证明Cdc 42诱导的MLK-3同源二聚化导致MLK-3的自磷酸化和活化，并假设自磷酸化是MLK-3活化的中间步骤，在这份报告中，我们试图进一步完善MLK-3活化的机制，并研究推定的激酶活化环在MLK-3活化中的作用。首先，我们将MLK-3激活环中的三个潜在磷酸化位点突变为丙氨酸，试图消除MLK-3的自磷酸化，突变体T277 A几乎不显示自磷酸化活性，几乎无功能;突变体S281 A显示低水平的自磷酸化，仅轻微激活其下游靶点，而T278 A突变体，显示出与野生型相当的自磷酸化的，几乎是完全功能性的。因此，活化环内的这些残基对于MLK-3自磷酸化和活化至关重要。此外，当Thr（277）和Ser（281）残基突变为带负电荷的谷氨酸以模拟磷酸化的丝氨酸/苏氨酸残基时，所得的突变体是完全功能性的，这意味着这两个残基可以作为自磷酸化位点。有趣的是，HPK 1也在体外磷酸化MLK-3激活环，并且发现Ser 281是主要的磷酸化位点，表明HPK 1也通过激酶激活环的磷酸化激活MLK-3。
We have demonstrated previously that Cdc42 induced MLK-3 homodimerization leads to both autophosphorylation and activation of MLK-3 and postulated that auto phosphorylation is an intermediate step of MLK-3 activation following its dimerization, In this report we sought to refine further the mechanism of MLK-3 activation and study the role of the putative kinase activation loop in MLK-3 activation. First we mutated the three potential phosphorylation sites in MLK-3 putative activation loop to alanine in an effort to abrogate MLK-3 autophosphorylation, Mutant T277A displayed almost no autophosphorylation activity and was nearly nonfunctional; mutant S281A, that displayed a low level of autophosphorylation, only slightly activated its downstream targets, whereas the T278A mutant, that exhibited autophosphorylation comparable to that of the wild type, was almost fully functional. Thus, these residues within the activation loop are critical for MLK-3 autophosphorylation and activation. In addition, when the Thr(277) and Ser(281) residues were mutated to negatively charged glutamic acid to mimic phosphorylated serine/threonine residues, the resulting mutants were fully functional, implying that these two residues may serve as the autophosphorylation sites. Interestingly, HPK1 also phosphorylated MLK-3 activation loop in vitro, and Ser281 was found to be the major phosphorylation site, indicating that HPK1 also activates MLK-3 via phosphorylation of the kinase activation loop.