Wang Lixin, Cummings Rhett, Zhao Yutong, Kazlauskas Andrius, Sham James K S, Morris Andrew, Georas Steve, Brindley David N, Natarajan Viswanathan
Department of Medicine, Division of Pulmonary and Critical Care, Johns Hopkins University, Baltimore, Maryland 21224, USA.
J Biol Chem. 2003 Oct 10;278(41):39931-40. doi: 10.1074/jbc.M302896200. Epub 2003 Jul 29.
Lysophosphatidate (LPA) mediates multiple cellular responses via heterotrimeric G protein coupled LPA-1, LPA-2, and LPA-3 receptors. Many G protein-coupled receptors stimulate ERK following tyrosine phosphorylation of growth factor receptors; however, the mechanism(s) of transactivation of receptor tyrosine kinases are not well defined. Here, we provide evidence for the involvement of phospholipase D (PLD) in LPA-mediated transactivation of platelet-derived growth factor receptor-beta (PDGF-R beta). In primary cultures of human bronchial epithelial cells (HBEpCs), LPA stimulated tyrosine phosphorylation of PDGF-R beta and threonine/tyrosine phosphorylation of ERK1/2. The LPA-mediated activation of ERK and tyrosine phosphorylation of PDGF-R beta was attenuated by tyrphostin AG 1296, an inhibitor of PDGF-R kinase, suggesting transactivation of PDGF-R by LPA. Furthermore, LPA-, but not PDGF beta-chain homodimer-induced tyrosine phosphorylation of PDGF-R beta was partially blocked by pertussis toxin, indicating coupling of LPA-R(s) to Gi. Exposure of HBEpCs to LPA activated PLD. Butan-1-ol, which acts as an acceptor of phosphatidate generated by the PLD pathway, blocked LPA-mediated transactivation of PDGF-R beta. This effect was not seen with butan-3-ol, suggesting PLD involvement. The role of PLD1 and PLD2 in the PDGF-R beta transactivation by LPA was investigated by infection of cells with adenoviral constructs of wild type and catalytically inactive mutants of PLD. LPA activated both PLD1 and PLD2 in HBEpCs; however, infection of cells with cDNA for wild type PLD2, but not PLD1, increased the tyrosine phosphorylation of PDGF-R beta in response to LPA. Also, the LPA-mediated tyrosine phosphorylation of PDGF-R beta was attenuated by the catalytically inactive mutant mPLD2-K758R. Infection of HBEpCs with adenoviral constructs of wild type hPLD1, mPLD2, and the inactive mutants of hPLD1 and mPLD2 resulted in association of PLD2 wild type and inactive mutant proteins with the PDGF-R beta compared with PLD1. These results show for the first time that transactivation of PDGF-R beta by LPA in HBEpCs is regulated by PLD2.
溶血磷脂酸（LPA）通过异源三聚体G蛋白偶联的LPA-1、LPA-2和LPA-3受体介导多种细胞反应。许多G蛋白偶联受体在生长因子受体酪氨酸磷酸化后刺激细胞外信号调节激酶（ERK）；然而，受体酪氨酸激酶反式激活的机制尚未完全明确。在此，我们提供证据表明磷脂酶D（PLD）参与LPA介导的血小板衍生生长因子受体β（PDGF-Rβ）的反式激活。在人支气管上皮细胞（HBEpC）的原代培养物中，LPA刺激PDGF-Rβ的酪氨酸磷酸化以及ERK1/2的苏氨酸/酪氨酸磷酸化。PDGF-R激酶抑制剂 tyrphostin AG 1296减弱了LPA介导的ERK激活和PDGF-Rβ的酪氨酸磷酸化，提示LPA对PDGF-R的反式激活作用。此外，百日咳毒素部分阻断了LPA而非PDGFβ链同二聚体诱导的PDGF-Rβ酪氨酸磷酸化，表明LPA受体与Gi偶联。将HBEpC暴露于LPA可激活PLD。作为PLD途径产生的磷脂酸受体的丁醇，阻断了LPA介导的PDGF-Rβ反式激活。丁醇未出现此效应，提示PLD参与其中。通过用野生型和催化失活突变体的腺病毒构建体感染细胞，研究了PLD1和PLD2在LPA对PDGF-Rβ反式激活中的作用。LPA激活HBEpC中的PLD1和PLD2；然而，用野生型PLD2而非PLD1的cDNA感染细胞，可增加LPA刺激下PDGF-Rβ的酪氨酸磷酸化。此外，催化失活突变体mPLD2-K758R减弱了LPA介导的PDGF-Rβ酪氨酸磷酸化。用野生型hPLD1、mPLD2以及hPLD1和mPLD2的失活突变体的腺病毒构建体感染HBEpC，结果显示与PLD1相比，PLD2野生型和失活突变体蛋白与PDGF-Rβ相关。这些结果首次表明，HBEpC中LPA对PDGF-Rβ的反式激活受PLD2调控。
