DeMali K A, Whiteford C C, Ulug E T, Kazlauskas A
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Biol Chem. 1997 Apr 4;272(14):9011-8. doi: 10.1074/jbc.272.14.9011.
Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.
尽管受体酪氨酸激酶的组成性激活会导致细胞转化这一点已得到充分证实，但其中涉及的信号转导途径尚未得到系统研究。在本研究中，我们使用了一组血小板衍生生长因子（PDGF）β受体突变体（β-PDGFR），这些突变体选择性地激活各种信号转导酶，以确定在软琼脂中PDGF依赖的细胞生长需要哪些信号通路。这些研究的宿主细胞系是Ph细胞，一种类似3T3的细胞，其表达正常水平的β-PDGFR，但不表达PDGF-α受体（α-PDGFR）。因此，该细胞系统可用于研究突变型αPDGFR或α/β嵌合体的信号传导。我们构建了包含αPDGFR细胞外结构域和具有各种磷酸化位点突变的βPDGFR细胞质结构域的嵌合受体。这些突变体在Ph细胞中表达，并检测它们驱动PDGF依赖的细胞转化（在软琼脂中生长）的能力。用空表达载体感染的细胞在软琼脂中无法生长，而引入具有野生型β-PDGFR细胞质结构域的嵌合体则产生了大量菌落。相比之下，N2F5嵌合体中磷脂酶Cγ（PLC-γ）、RasGTP酶激活蛋白、磷脂酰肌醇3激酶（PI3K）和SHP-2的结合位点被消除，未能触发增殖。恢复RasGTP酶激活蛋白或SHP-2的结合位点并不能挽救PDGF依赖的反应。相反，能够与PLC-γ或PI3K结合的受体在软琼脂生长试验中传递的生长信号与野生型受体相当。这些发现表明，PDGF受体激活多种导致细胞转化的信号通路，并且PI3K或PLC-γ是此类信号转导级联反应的关键启动因子。
