Hordijk P L, Verlaan I, van Corven E J, Moolenaar W H
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
J Biol Chem. 1994 Jan 7;269(1):645-51.
Lysophosphatidic acid (LPA) is a platelet-derived phospholipid that serves as a mitogen for fibroblasts. LPA activates its own G protein-coupled receptor(s) leading to stimulation of phospholipase C and inhibition of adenylate cyclase. Furthermore, LPA rapidly activates p21ras through a pertussis toxin-sensitive pathway. In this study, we have examined LPA-induced protein tyrosine phosphorylation in Rat-1 fibroblasts. LPA action was compared with that of endothelin, which is a stronger activator of phospholipase C than LPA but fails to activate p21ras and to stimulate DNA synthesis in these cells. LPA and, more effectively, endothelin rapidly stimulate tyrosine phosphorylation of proteins of 110-130, 95, and 65-75 kDa. The effect of LPA is dose- and time-dependent, being half-maximal at 3-30 nM and peaking after 2-5 min. Among the 110-130-kDa group of phosphotyrosyl proteins is the 125-kDa "focal adhesion kinase" (p125FAK) but not the 120-kDa p21ras GTPase-activating protein. Furthermore, LPA, like epidermal growth factor, causes tyrosine phosphorylation and activation of the p42/p44 mitogen-activated protein (MAP) kinases, paralleling p21ras activation. In contrast, endothelin fails to phosphorylate MAP kinase. Treatment of the cells with pertussis toxin blocks LPA-induced MAP kinase phosphorylation without affecting the other tyrosine phosphorylations. The kinase inhibitor staurosporine (1 microM) blocks LPA-induced, but not epidermal growth factor-induced, activation of p21ras and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21ras activation. The results support a model in which LPA-induced phosphorylation of MAP kinase is mediated by p21ras, and tyrosine phosphorylation of the other substrates, including p125FAK, is associated with phospholipase C activation.
溶血磷脂酸（LPA）是一种血小板衍生的磷脂，可作为成纤维细胞的促有丝分裂原。LPA激活其自身的G蛋白偶联受体，导致磷脂酶C的刺激和腺苷酸环化酶的抑制。此外，LPA通过百日咳毒素敏感途径快速激活p21ras。在本研究中，我们检测了LPA诱导的大鼠-1成纤维细胞中的蛋白质酪氨酸磷酸化。将LPA的作用与内皮素的作用进行了比较，内皮素是比LPA更强的磷脂酶C激活剂，但不能激活p21ras，也不能刺激这些细胞中的DNA合成。LPA以及更有效的内皮素能快速刺激110 - 130 kDa、95 kDa和65 - 75 kDa蛋白质的酪氨酸磷酸化。LPA的作用具有剂量和时间依赖性，在3 - 30 nM时达到半数最大效应，在2 - 5分钟后达到峰值。在110 - 130 kDa的磷酸酪氨酸蛋白组中是125 kDa的“粘着斑激酶”（p125FAK），而不是120 kDa的p21ras GTP酶激活蛋白。此外，LPA与表皮生长因子一样，导致酪氨酸磷酸化并激活p42/p44丝裂原活化蛋白（MAP）激酶，与p21ras激活平行。相比之下，内皮素不能使MAP激酶磷酸化。用百日咳毒素处理细胞可阻断LPA诱导的MAP激酶磷酸化，而不影响其他酪氨酸磷酸化。激酶抑制剂星形孢菌素（1 microM）可阻断LPA诱导的但不是表皮生长因子诱导的p21ras和MAP激酶的激活，这与一种连接LPA受体与p21ras激活的中间蛋白激酶一致。结果支持了一个模型，其中LPA诱导的MAP激酶磷酸化由p21ras介导，而其他底物（包括p125FAK）的酪氨酸磷酸化与磷脂酶C激活相关。
