Abstract
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.
Highlights
Lysophosphatidic acid (LPA) is a platelet-derived wide range of biological responses in diverse cell types, apparphospholipid that serves as a mitogen for fibroblasts. ently by activating its cognate G protein-coupled receptor(s)
LPA action wascompared with thatof endothelin, which is a stronger activator of phospholipase C than LPA but fails to activatep21'" and to stimulate DNA synthesis in these cells
The kinase inhibitor play a central role in the stimulation of DNA synthesis by staurosporine (1PM) blocks LPA-induced, but not epidermal growth factor-induced, activatiofn p21" and MAP kinase, consistent with an intermediate protein kinase linking the LPA receptor to p21'" activation
Summary
Gene Pharming, 2333 CC Leiden, The Netherreorganization of the actin cytoskeleton and subsequent retraction of developing neurites (4). Phosphorylation and Activation of MAP Kinase-The antipTyr immunoblotting experiments reveal that LPA and EGF, but not endothelin, induce tyrosine phosphorylation of two proteins of[42] and 44 kDa (Fig. l),which are likely to represent the ~ 4 2 1 ~ 4M4AP kinases. Theseare activated through phosphorylation on Tyr and Thrersidues in response to various external signals including peptide growth factors, phorbol ester, and G protein-coupled receptor agonists (2025, 41-43) This activation is readily detectable on anti-MAP kinase immunoblots, where the activated, phosphorylated forms of ~ 4 2 1 ~ d4i4splay a lower electrophoretic mobility (29, 44).Fig. 5 (upper panel) shows that both LPA andEGF induce a rapid shift in mobility of the p42/p44 MAP kinases. Stimulation of Rat-1 cells with either LPA or EGF results in enhanced phosphorylation of myelin basic proteinin MAP kinase immunoprecipitates (Fig.[5], lower panel), showing that MAP kinase phosphorylation parallels increased enzymatic activity.
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