Rankin S, Rozengurt E
Imperial Cancer Research Fund, London, United Kingdom.
J Biol Chem. 1994 Jan 7;269(1):704-10.
In the present study, we have identified several proteins in Swiss 3T3 cells that are phosphorylated on tyrosine in response to platelet-derived growth factor (PDGF) and exhibit an unusual bell-shaped dose-response curve with a maximum at 5 ng/ml platelet-derived growth factor (PDGF). These proteins include two that are associated with focal adhesions, namely the focal adhesion kinase (p125FAK), a novel cytosolic tyrosine kinase, and paxillin. At low concentrations of PDGF (1-5 ng/ml), these proteins are the predominant tyrosine-phosphorylated species. At 30 ng/ml PDGF, however, there was no stimulation of their phosphorylation over control levels. In contrast, tyrosine phosphorylation of previously described substrates of the PDGF receptor tyrosine kinase, namely the p21ras GTPase-activating protein, p120, phosphatidyl inositol 3' kinase, and phospholipase C gamma exhibited sigmoidal dose-response curves with PDGF and were all efficiently phosphorylated on tyrosine at 30 ng/ml PDGF. Cytochalasin D, which disrupts the actin cytoskeleton, completely inhibited the tyrosine phosphorylation of p125FAK and paxillin by PDGF. Examination of the actin cytoskeleton after stimulation of cells with different concentrations of PDGF revealed that at 5 ng/ml PDGF, actin appears in stress fibers and in membrane ruffles, while at 30 ng/ml, PDGF disrupts the actin cytoskeleton. Bombesin stimulates actin stress fiber formation with no evidence of disruption of stress fibers at high concentrations. When cells were stimulated with bombesin (10 nM) in the presence of 30 ng/ml PDGF, however, the actin cytoskeleton was completely disrupted. Further, the tyrosine phosphorylation of both p125FAK and paxillin induced by bombesin (10 nM) was completely prevented when cells were stimulated with bombesin in the presence of 30 ng/ml PDGF. We propose that the inhibitory limb in the bell-shaped dose-response curve of PDGF and the novel cross-talk between PDGF and bombesin on tyrosine phosphorylation may be explained by the ability of PDGF at 30 ng/ml to disrupt the actin cytoskeleton.
在本研究中，我们在瑞士3T3细胞中鉴定出了几种蛋白质，它们在受到血小板衍生生长因子（PDGF）刺激时会发生酪氨酸磷酸化，并呈现出异常的钟形剂量反应曲线，在5 ng/ml血小板衍生生长因子（PDGF）时达到最大值。这些蛋白质包括两种与粘着斑相关的蛋白质，即粘着斑激酶（p125FAK），一种新的胞质酪氨酸激酶，和桩蛋白。在低浓度的PDGF（1 - 5 ng/ml）下，这些蛋白质是主要的酪氨酸磷酸化物种。然而，在30 ng/ml PDGF时，它们的磷酸化水平并没有超过对照水平。相比之下，PDGF受体酪氨酸激酶先前描述的底物，即p21ras GTP酶激活蛋白、p120、磷脂酰肌醇3'激酶和磷脂酶Cγ的酪氨酸磷酸化呈现出与PDGF的S形剂量反应曲线，并且在30 ng/ml PDGF时酪氨酸都能有效地磷酸化。细胞松弛素D可破坏肌动蛋白细胞骨架，它完全抑制了PDGF对p125FAK和桩蛋白的酪氨酸磷酸化。用不同浓度的PDGF刺激细胞后检查肌动蛋白细胞骨架发现，在5 ng/ml PDGF时，肌动蛋白出现在应力纤维和膜皱褶中，而在30 ng/ml时，PDGF破坏了肌动蛋白细胞骨架。蛙皮素刺激肌动蛋白应力纤维形成，在高浓度时没有应力纤维破坏的迹象。然而，当细胞在30 ng/ml PDGF存在的情况下用蛙皮素（10 nM）刺激时，肌动蛋白细胞骨架被完全破坏。此外，当细胞在30 ng/ml PDGF存在的情况下用蛙皮素刺激时，蛙皮素（10 nM）诱导的p125FAK和桩蛋白的酪氨酸磷酸化被完全抑制。我们提出，PDGF钟形剂量反应曲线中的抑制部分以及PDGF和蛙皮素在酪氨酸磷酸化上的新型相互作用，可能是由30 ng/ml的PDGF破坏肌动蛋白细胞骨架的能力所解释的。
