Figure 15–48 Transient activation of Ras revealed by single-molecule fluorescence resonance energy transfer (FReT). (A) Schematic drawing of the experimental strategy. Cells of a human cancer cell line are genetically engineered to express a Ras protein that is covalently linked to yellow fluorescent protein (YFP). GTP that is labeled with a red fluorescent dye is microinjected into some of the cells. The cells are then stimulated with the extracellular signal protein EGF, and single fluorescent molecules of Ras-YFP at the inner surface of the plasma membrane are followed by video fluorescence microscopy in individual cells. When a fluorescent Ras-YFP molecule becomes activated, it exchanges unlabeled GDP for fluorescently labeled GTP; the energy emitted by the YFP now activates the fluorescent GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence from a previously yellow-green fluorescent spot at the plasma membrane. As shown in (B), activated Ras molecules can be detected after about 30 seconds of EGF stimulation. The red signal peaks at about 3 minutes and then decreases to baseline by 6 minutes. As Ras-GAP is found to be recruited to the same spots at the plasma membrane as Ras, it presumably plays a major part in rapidly shutting off the Ras signal. (Modified from H. Murakoshi et al., Proc. Natl Acad. Sci. USA 101:7317–7322, 2004. With permission from National Academy of Sciences.)
Ras to inactivate itself by hydrolyzing its bound GTP to GDP. To stimulate cells to proliferate or differentiate, these short-lived signaling events must be converted into longer-lasting ones that can sustain the signal and relay it downstream to the nucleus to alter the pattern of gene expression. One of the key mechanisms used for this purpose is a system of proteins called the mitogen-activated protein kinase module (MAP kinase module) (Figure 15–49). The three components of this system form a functional signaling module that has been remarkably well conserved during evolution and is used, with variations, in many different signaling contexts.
The three components are all protein kinases. The final kinase in the series is called simply MAP kinase (MAPK). The next one upstream from this is MAP kinase kinase (MAPKK): it phosphorylates and thereby activates MAP kinase. Next above that, receiving an activating signal directly from Ras, is MAP kinase kinase kinase (MAPKKK): it phosphorylates and thereby activates MAPKK. In the mammalian Ras–MAP-kinase signaling pathway, these three kinases are known by shorter names: Raf (= MAPKKK), Mek (= MAPKK), and Erk (=MAPK).
Once activated, the MAP kinase relays the signal downstream by phosphorylating various proteins in the cell, including transcription regulators and other protein kinases (see Figure 15–49). Erk, for example, enters the nucleus and phosphorylates one or more components of a transcription regulatory complex. This activates the transcription of a set of immediate early genes, so named because they turn on within minutes after an RTK receives an extracellular signal, even if protein synthesis is experimentally blocked with drugs. Some of these genes encode other transcription regulators that turn on other genes, a process that requires both protein synthesis and more time. In this way, the Ras–MAP-kinase signaling pathway conveys signals from the cell surface to the nucleus and alters the pattern of gene expression. Among the genes activated by this pathway are some that stimulate cell proliferation, such as the genes encoding G1 cyclins (discussed in Chapter 17).