Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay
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DOI：
10.1371/journal.pone.0062871
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发表时间：
2013-05-01
期刊：
影响因子：
3.7
通讯作者：
Bohmer, Frank-D.
中科院分区：
文献类型：
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作者：
Bohmer, Sylvia-Annette;Weibrecht, Irene;Bohmer, Frank-D.
蛋白质酪氨酸磷酸酶（Protein-tyrosine phosphatases，PTPs）是信号转导过程的重要调节因子。必不可少的PTP的功能表征是其生理底物的识别，而实现这一目标的重要一步是物理相互作用的演示。然而，PTP与其细胞底物的结合通常是短暂的，并且难以在内源性水平上用未修饰的蛋白质检测。密度增强型磷酸酶-1（DEP-1/PTPRJ）是造血细胞功能的调节因子，也是一种候选的肿瘤抑制因子。然而，协会DEP-1与任何其提出的基板在内源性水平尚未显示。我们以前已经获得了功能和生物化学的证据DEP-1与造血受体酪氨酸激酶Fms样酪氨酸激酶3（FLT 3）的直接相互作用。在目前的研究中，我们已经使用的方法，在原位邻近连接试验（原位PLA），以验证这种相互作用在内源性水平，并进一步表征it. In原位PLA容易检测到协会的内源性DEP 1和FLT 3在人急性单核细胞白血病细胞系THP-1，这是增强FLT 3配体（FL）的刺激在一个时间依赖性的方式。协会达到高峰之间的10和20分钟的刺激，并返回到基础水平在30分钟。这个时间过程是类似的FLT 3自磷酸化的时间过程。FLT 3激酶抑制和DEP-1氧化消除关联。与DEP-1-FLT 3相互作用的功能作用一致，THP-1细胞中DEP-1的稳定敲除增强FL诱导的ERK 1/2活化。这些发现支持FLT 3是DEP-1的真正底物，并且相互作用主要通过FLT 3配体刺激触发的酶-底物复合物形成而发生。
Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation.