Zhixiang Wang

Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival.

ABSTRACT In spite of intensified efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways and no evidence to demonstrate that endosomal signaling is able to produce a biological outcome. The lack of breakthrough is due in part to the lack of means to generate endosomal signals without plasma membrane signaling. In this paper, we report the establishment of a system to specifically activate epidermal growth factor (EGF) receptor (EGFR) when it endocytoses into endosomes. We treated cells with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks the recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complexes into endosomes. The endosome-associated EGFR was then activated by removing AG-1478 and monensin. During this procedure we did not observe any surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. By using this system, we provided original evidence demonstrating that (i) the endosome can serve as a nucleation site for the formation of signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal.

Grb2 and Shc Adapter Proteins Play Distinct Roles in Neu (ErbB-2)-Induced Mammary Tumorigenesis: Implications for Human Breast Cancer

ABSTRACT Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.

Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF‐induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.

Platelet-derived growth factor receptor-mediated signal transduction from endosomes

Although accumulated evidence supports the concept of endosomal signaling of receptor tyrosine kinases, most results are generated from studies of epidermal growth factor receptor (EGFR). It is not clear whether the concept of endosomal signaling could be generally applied to the other receptor tyrosine kinases. For example, platelet-derived growth factor receptor (PDGFR) is very similar to EGFR in terms of both signaling and trafficking; however, little is known about the endosomal signaling of PDGFR. In this research, we applied the same approaches from our recent studies regarding EGFR endosomal signaling to investigate the endosomal signaling of PDGFR. We showed in this communication that we are able to establish a system that allows the specific activation of endosome-associated PDGFR without the activation of the plasma membrane-associated PDGFR and without disrupting the overall endocytosis pathway. By using this system, we showed that endosomal activation of PDGFR recruits various signaling proteins including Grb2, SHC, phospholipase C-γ1, and the p85α subunit of phosphatidylinositol 3-kinase into endosomes and forms signaling complexes with PDGFR. We also showed that endosomal PDGFR signaling is sufficient to activate the major signaling pathways implicated in cell proliferation and survival. Moreover, we demonstrate that endosomal PDGFR signaling is sufficient to generate physiological output including cell proliferation and cell survival.

Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling

ABSTRACT Strong evidence indicates that endosome-localized epidermal growth factor receptor (EGFR) plays an important role in cell signaling. However, elimination of endosomal signaling does not attenuate EGF-induced physiological outcomes, arguing against physiological relevance. Recently we established a system to specifically activate endosome-associated EGFR in the absence of any plasma membrane activation of EGFR and showed that endosomal EGFR signaling is sufficient to support cell survival. However, this pure endosomal signaling of EGFR does not stimulate cell proliferation, because EGFR only remained activated for less than 2 h following its stimulation at endosomes, while DNA synthesis generally requires growth factor exposure for 8 h or more. Here we report that the prolonged requirement for EGF to stimulate epithelial cell proliferation can be substituted for with two short pulses of EGF. By combining the two short pulses of EGF stimulation with our previously established method to generate endosomal EGFR signaling, we are able to generate two pulses of endosomal EGFR signaling. In this way, we demonstrated that two pulses of endosomal EGFR signaling are sufficient to stimulate cell proliferation. The first pulse of EGFR signaling induces exit from quiescence into G1 phase and appears to render cells responsive to subsequent mitogenic stimulus. This second pulse, required several hours later, drives cells through the restriction point of late G1 and into S phase. We further showed that the two pulses of endosomal EGFR signaling engaged cell cycle machinery the same way as the two pulses of standard EGFR signaling. Moreover, two pulses of endosomal EGFR signaling stimulated downstream signaling cascades in a similar way to the two pulses of standard EGFR activation. The data therefore demonstrate that signals transduced from internalized EGFR, with or without a contribution from the plasma membrane, fully satisfy the physiological requirements for S-phase entry.

A Tale of Two Cbls: Interplay of c-Cbl and Cbl-b in Epidermal Growth Factor Receptor Downregulation

ABSTRACT The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.

PLC-γ1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell Migration

It is well established that epidermal growth factor (EGF) induces the cytoskeleton reorganization and cell migration through two major signaling cascades: phospholipase C-gamma1 (PLC-gamma1) and Rho GTPases. However, little is known about the cross talk between PLC-gamma1 and Rho GTPases. Here we showed that PLC-gamma1 forms a complex with Rac1 in response to EGF. This interaction is direct and mediated by PLC-gamma1 Src homology 3 (SH3) domain and Rac1 (106)PNTP(109) motif. This interaction is critical for EGF-induced Rac1 activation in vivo, and PLC-gamma1 SH3 domain is actually a potent and specific Rac1 guanine nucleotide exchange factor in vitro. We have also demonstrated that the interaction between PLC-gamma1 SH3 domain and Rac1 play a significant role in EGF-induced F-actin formation and cell migration. We conclude that PLC-gamma1 and Rac1 coregulate EGF-induced cell cytoskeleton remodeling and cell migration by a direct functional interaction.

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3‐kinase (PI3K) have been proposed to co‐regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)‐stimulated PI3K activity by expression of the kinase‐deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR‐containing endosomes and dissociates the early‐endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti‐Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co‐regulated by Rab5 and PI3K.

Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase

The involvement of phosphatidylinositol 3‐kinase (PI3K) in membrane trafficking in mammalian cells has largely come from experiments with wortmannin. This compound inhibits endosome fusion in vitro, possibly by inhibiting the production of phosphatidylinositol (PtdIns)‐3‐P, which co‐regulates EEA1 with Rab5. However, the results from wortmannin inhibition experiments performed in vivo differ significantly. We have recently shown that wortmannin enlarges endosomes containing the epidermal growth factor receptor (EGFR) and enhances the lysosomal degradation of EGFR. In this report, we demonstrate that addition of the PI3K reaction products does not suppress wortmannin‐induced enlargement of EGFR‐containing endosomes and enhancement of EGFR degradation. Moreover, the effects of wortmannin on the intracellular trafficking of EGFR mimic those of the permanently activated Rab5 mutant, Rab5 Q79L, which stimulates endosome fusion. We also found that an inactive Rab5 mutant, Rab5 S34N, blocks wortmannin‐induced endosome enlargement and that wortmannin stimulates the activation of Rab5. We further showed that wortmannin reduced the membrane association of p120 Ras GTPase‐activating protein (GAP) and inhibited the interaction between Rab5 and p120 Ras GAP. We conclude that wortmannin alters intracellular trafficking of EGFR by activating Rab5 rather than by inhibiting PI3K.

Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer. Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation. These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways. These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle. Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression. We focus on the induction of CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways. We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors.