Copyright notice. This article has been cited by other articles in PMC. Open in a separate window. Mechanistic Insight. Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank Maojing Yang for excellent technical assistance. Footnotes The authors declare no conflict of interest. References 1. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB.
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J Biol Chem. Chen D, et al. Mol Cell Biol. Tanaka K, et al. Cancer Discov. Jiang T, et al. Cancer Res. Guo C, Stark GR. Buday L, Downward J. Many faces of Ras activation. Biochim Biophys Acta. Jorge R, et al. HSos1 contains a new amino-terminal regulatory motif with specific binding affinity for its pleckstrin homology domain. When expressed individually or coexpressed, both H-Ras and K-Ras were frequently seen in the endosomal compartments, although the extent of accumulation of H-Ras in endosomes was relatively higher than that of K-Ras.
It is possible that H-Ras is delivered to endosomes by means of vesicular transport from the Golgi apparatus. In addition, as revealed by time-lapse imaging, H-Ras can be internalized from the plasma membrane Figure 6.
The internalization of GFP-H-Ras in insulin-stimulated Rat-1 cells was recently demonstrated by evanescent wave microscopy Rizzo et al. The ability of H-Ras to traffic through endosomes may play an important role in Rab5 activation, which leads to extensive endosome fusion and formation of large endosomes Tall et al. Correct trafficking of Ras does not seem to require GTP binding because the distribution of the H-RasN17 in the cells was essentially similar to that of wild-type H-Ras.
However, expression of N17 mutant did not result in endosome fusion Figure 4 , suggesting that GTP loading of Ras is required for Rab5 activation. Previous studies of Ras localization defined the plasma membrane as the main cellular location of Ras Willingham et al. More recent studies explored Ras microdomain compartmentalization within the plasma membrane Prior et al. These previous results do not contradict our observations. Although a significant amount of transfected H-Ras and K-Ras was seen in the intracellular membranes, a larger pool of Ras fusion proteins was associated with the plasma membrane, particularly, in the areas with ruffling activity.
However, previous studies and this work Figure 3 showed that endogenous Ras is also detected in the membrane compartments in the Golgi area Choy et al. Thus, it can be proposed that trafficking of tagged Ras proteins reflects the behavior of endogenous Ras, although the intensity of trafficking through some pathways may be exaggerated. This assay, however, relies on the expression of tagged Ras.
The overexpression of H-Ras leads to activation of a substantial pool of H-Ras regardless of the conditions of serum starving or stimulation with EGF. Significant activity of heterologously expressed Ras in serum-starved cells has also been reported by others Lockyer et al.
The existence of a large pool of GTP-bound but effector-inaccessible endogenous Ras has been demonstrated Hamilton et al. Therefore, this technique does not allow monitoring of the activation of total cellular Ras by EGFR, but rather, it is designed to analyze the movement of activated Ras within the cell. A recent study by Mochizuki et al. However, it is not clear whether the fatty acid modifications and cellular localization of the chimeric protein are similar to that of wild-type Ras.
A number of studies have used various technical approaches to explore the hypothesis of endosomal signaling by EGFRs reviewed in Baass et al. These approaches mainly included the isolation of endosomes, biochemical separation of internalized from surface EGFRs, and immunohistochemical studies in fixed cells Lai et al. The requirement of EGFR endocytosis for activation of mitogen-activated protein kinase was revealed in cells in which the endocytosis was inhibited by mutant dynamin Vietra et al.
One type of information that FRET microscopy can offer is a quantitative description of the cellular distribution of interacting complexes and the extent of interaction. In contrast, coimmunoprecipitation experiments yield only very low amounts of Grb2 and Shc associated with the EGFR, probably due to instability of complexes in detergent solutions Di Gugliemo et al. Consequently, our results imply that Grb2—SOS complexes become unavailable to Ras located at the plasma membrane.
Thus, massive endocytosis of Grb2 may lead to physical proximity of Grb2—SOS complex to endosomal Ras at the expense of the separation of this complex from plasma membrane Ras and its effectors. In this case, preferential localization of H-Ras in endosomes may explain its greater capacity to activate PI3 kinase compared with that of K-Ras.
The demonstration of a protein—protein interaction in living cells is a critical test of hypotheses originating from biochemical or other experiments requiring cell disruption.
The ability to assign protein—protein interactions to specific compartments of the cell opens the way to more complete understanding of the regulation of signaling processes. For instance, in addition to regulation of output signals by Ras, Ras activity itself may be determined by its distribution. We thank Dr. Article published online ahead of print. Cell Article and publication date are at www.
Molecular Biology of the Cell Vol. This is the final version - click for previous version. Xuejun Jiang Search for more papers by this author.
E-mail address: E-mail Address: alexander. Search for more papers by this author. Add to favorites Download Citations Track Citations. View article. Abstract Activation of the epidermal growth factor receptor EGFR triggers multiple signaling pathways and rapid endocytosis of the epidermal growth factor EGF —receptor complexes. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Compartmentalized signal transduction by receptor tyrosine kinases.
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