Carey Kendall D, Watson Robert T, Pessin Jeffrey E, Stork Philip J S
Vollum Institute, Department of Cell and Developmental Biology, L474 Oregon Health Sciences University, Portland, Oregon 97201, USA.
J Biol Chem. 2003 Jan 31;278(5):3185-96. doi: 10.1074/jbc.M207014200. Epub 2002 Nov 21.
Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.
Ras对Raf-1的激活需要募集到细胞膜以及进行额外的磷酸化，包括丝氨酸338（Ser-338）和酪氨酸341（Tyr-341）位点的磷酸化。在本研究中，我们发现酪氨酸341参与了Raf-1募集到称为“脂筏”的特殊膜结构域，而这是Raf-1在丝氨酸338位点发生磷酸化所必需的。Raf-1也被认为在Rap1加载GTP后被募集到小G蛋白Rap1上。然而，这并不会导致Raf-1的激活。我们认为这是因为Raf-1在募集到Rap1时酪氨酸341位点未发生磷酸化。将Rap1重定向到含有Ras的细胞膜上，或者通过突变模拟Raf-1的酪氨酸341磷酸化，可将Rap1转化为Raf-1的激活剂。与Raf-1不同，B-Raf可被Rap1激活。我们认为这是因为B-Raf的激活不依赖于酪氨酸磷酸化。此外，使B-Raf依赖于酪氨酸磷酸化的突变体不再被Rap1激活。
