H-Ras Signaling and K-Ras Signaling Are Differentially Dependent on Endocytosis

Abstract

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras-but not K-Rasmediated Raf-1 activation in BHK cells. H-Ras-but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…[8] More recently, thanks to the pioneering work of M. Philips, it was demonstrated that Ras was also present and active in endomembranes like the endoplasmic reticulum (ER) and the Golgi complex (GC). [9,10] The three Ras isoforms were also detected in endosomes [11,12] and mitochondria [13][14][15] (for a review see ref. 16).…”

“…[29] Interestingly, Ras association to endosomes exhibits isoform specificity: whereas N-and H-Ras establish stable interactions with the endosomal compartment, K-Ras does not. [11,30,31] These differences have been attributed to the modification of N-and H-Ras, but not of K-Ras, by ubiquitination. [31] At the PM, Ras proteins partition into microdomains referred to as ''nanoclusters.''…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Growth factor receptor endocytosis is necessary for efficient activation of the Raf-MAPK cascade and promotion of differentiation versus proliferation [104]. Isoform-specific signalling was seen when inhibition of dynamin dependent endocytosis selectively inhibited oncogenic H-Ras but not K-Ras activation of Raf [69]. Mitochondria have also been identified as sites of Ras activation.…”

Differential Regulation of RasGAPs in Cancer

“…[57][58][59] The fact that p120GAP remains associated with EGFR in endosomes 60 is another indication for the involvement of GAPs in the regulation of spatiotemporal Ras signaling, as active H-Ras is internalized with EGFR after stimulation. 45 The targeting protein for p120GAP, AnxA6, is also found in endosomes and able to bind active H-Ras. 30,39,40 Altogether, this could ensure H-Ras signal termination both at the plasma membrane and in early endosomes upon EGFR activation.…”

“…NRas-GTP is mainly found in cholesterolsensitive membrane domains. It was initially believed that Ras signaling occurs exclusively at the plasma membrane, but since the discovery of active Ras in the endoplasmic reticulum and Golgi complex, 43,44 all 3 Ras isoforms have been detected in endosomes [45][46][47] and mitochondria. [48][49][50] Thus, Ras isoforms cannot be considered stationary but translocate between cellular compartments.…”