Molecular Mechanism And Biological Significance Of SGEF Enhancing The Stability Of EGFR Protein

Aberrant activation of EGFR signaling correlates with high grade and advancedstages of cancer, and a high risk for recurrence and progression to hormone refractorystatus. Studies on a series of prostate adenocarcinomas revealed that EGFRoverexpression was the most frequent EGFR-related abnormality (31%) while EGFRmutation was a minor event (only8%). Moreover, there was no correlation betweengene copy number and EGFR protein expression suggesting that EGFR overexpressionin prostate cancer may be primarily due to the deregulation of EGFR signaling.Previously we demonstrated an elevated SGEF expression in clinical specimens withprostate cancer and implicated the role of SGEF in prostate tumorigenesis. However,the molecular mechanism behind the SGEF regulation of prostate cancer developmentis not known. One of our previous results showed that SGEF enhanced thephosphorylation of AKT in prostate cancer cells. Since EGFR is an important upstreamactivator of AKT, we speculated that SGEF may enhance the EGFR/AKT signaling byenhancing EGFR protein level.We first analyzed the effect of SGEF depletion on the total level of EGFR protein.The results showed that knockdown of SGEF in prostate cancer cells decreased EGFRprotein levels. This observation suggests the involvement of SGEF in EGFR turnover.To verify this presumption, we determined the effect of SGEF overexpression on EGFinduced EGFR degradation in HEK293T and COS-7cells. We found that EGFR proteindegradation is antagonized in cells overexpressing SGEF. To confirm the effect ofSGEF on EGFR stability in prostate cancer cells, we silenced SGEF expression in threeprostate cancer cell lines (C4-2, C4-2B and DU145) and determined EGFR proteinlevels after treating with EGF for varying periods of time. We showed that silencing theexpression of SGEF in prostate cancer cells accelerates EGFR degradation. These datasuggest that SGEF plays an important role in EGFR degradation. Since SGEF ischaracterized as a GEF for RhoG, we speculated that the negative effect of SGEF onEGFR degradation may depend on RhoG function. However, we found that deletion ofthe SGEF DH domain did not abrogate its effect on EGFR stability. Furthermore,constitutively active RhoG could not restore the accelerated degradation of EGFR inSGEF depleted DU145cells, suggesting that RhoG function is not essential for SGEF-mediated EGFR stability.After binding to EGF, EGFR undergoes dimerization which promotes auto-phosphorylation of multiple tyrosine residues in the cytoplasmic tail of the receptor. These phosphorylated EGFR recruits E3ubiquitin ligase Cbl, which promote EGFRubiquitination. Ubiquitination of EGFR initiates events leading to its internalization anddelivery to early endosomes, from where it is sorted to lysosomes for degradation. Wefound that the inhibitory role of SGEF on EGFR degradation is not attributed to itsnegative effects on EGFR ubiquitination and internalization. The results show thatSGEF overexpression has little effect on EGFR trafficking in early endosomes butinhibits the exit of EGFR from early endosomes and its transport to late endosomes.We further showed that depletion of SGEF promotes EGFR transit from early to lateendosomes, suggesting that SGEF inhibits EGFR degradation by delaying EGFRtrafficking from early to late endosomes.Overexpression of EGFR in general, is coupled with enhanced phosphorylation ofEGFR. Therefore, we analyzed the effect of SGEF on the phosphorylation of EGFRand its downstream effector AKT after simulation with EGF. The results show thatSGEF depletion significantly inhibited EGF-induced phosphorylation of EGFR andAKT in prostate cancer cells. Since AKT has been reported to play a pivotal role inEGFR-mediated prostate cancer cell migration, we further examined the effect of SGEFon EGFR-directed prostate cancer cell migration. We found that EGF-inducedmigration in SGEF depleted prostate cancer cells is profoundly attenuated compared tocontrol cells. These results integrate the effects of SGEF on EGFR stability, EGFR sig-naling and EGFR mediated cellular functions.EGF induced ERK1/2signaling plays important roles in numerous cellularactivities. Since we found that SGEF attenuates EGFR degradation and enhances EGFRstability, we investigated whether SGEF regulates EGF induced ERK1/2activation, amajor effector downstream of EGFR. Our results show that overexpression of SGEFenhanced EGF induced ERK1/2activation and silencing the expression of SGEFinhibited EGF induced ERK1/2activation, suggesting that SGEF enhances EGFinduced ERK1/2activation. To evaluate whether RhoG function is required for SGEFmediated EGFR/ERK1/2signaling, we suppressed RhoG expression in HEK293T cellsand examined the effects of SGEF overexpression on EGF-induced ERK1/2phosphorylation. We found that depletion of RhoG did not abrogate SGEF mediatedEGFR/ERK1/2activation, suggesting that RhoG function is not essential for SGEF toeffect EGF induced ERK1/2activation.The adaptor protein Grb2plays a critical role in EGF-activated Ras-ERK1/2signaling pathway. Grb2contains two SH3domains and SGEF contains a Pro domain in its N terminus. Since Pro domain is well-known to recognize SH3domains tomediate the interaction of proteins, we speculated that Grb2may interact with SGEF.Our results show that the interaction of SGEF with Grb2and the critical role of theGrb2SH3domains and the SGEF Pro domain in this interaction. Therefore, it isreasonable to speculate that this interaction is essential for the effect of SGEF on EGFinduced ERK1/2activation. Surprisingly, our results demonstrate that impairing theinteraction between Grb2and SGEF further improved EGF-induced ERK1/2activationrather than weakening it. This unexpected result suggests the possible antagonizing roleof Grb2on the enhancement of EGF induced ERK1/2activation by interacting withSGEF. Therefore, we analyzed the effect of Grb2overexpression or depletion on SGEFmediated EGFR/ERK1/2activation. The results show that Grb2overexpressionsuppresses SGEF mediated ERK1/2activation. In addition, Grb2depletionsignificantly enhances SGEF mediated ERK1/2activation. Moreover, our data furtherdemonstrate that the interaction of Grb2with SGEF is critical for this antagonistic effect.In summary, we report that SGEF enhances EGFR stability and signaling bydelaying its lysosomal sorting and degradation. Because excessive or inappropriateEGFR signaling is associated with malignant progression of prostate cancer, theregulation of EGFR stability by SGEF is likely to be important for the development ofprostate cancer. In addition, since degradation of various growth factor receptors aresimilar to EGFR degradation, SGEF may be implicated in the lysosomal sorting anddegradation of a series of receptors, thereby enhancing its oncogenic potential. We alsoshowed that SGEF enhances EGF induced ERK1/2activation independent of its GEFactivity. Further, our study has demonstrated that Grb2antagonizes the ability of SGEFto enhance EGF induced ERK1/2activation by interacting with SGEF, going beyondits canonical function as a critical downstream transducer for several growth factorreceptors, providing important insights into the complex role of Grb2in EGFR signaltransduction.