In control experiments, reduced phosphorylation of ERK1/2 was observed in EGF-stimulated HeLa/MEK2CGFP cells treated with UO126 (Number 9A). However, triggered MEK was recognized only in the plasma membrane and not in endosomes. Remarkably, MEK2CGFP endosomes did not contain active EGFR, suggesting that endosomal MEK2CGFP was separated from your upstream signaling complexes. Knockdown of clathrin by small interfering RNA (siRNA) abolished MEK2 recruitment to endosomes but resulted in improved activation of ERK without influencing the activity of MEK2CGFP. The build up of MEK2CGFP in endosomes was also clogged by siRNA depletion of RAF kinases and by the MEK1/2 inhibitor, UO126. We propose that the recruitment of MEK2 to endosomes can be a part of the bad feedback regulation of the EGFRCMAPK signaling pathway by endocytosis. = 2). D) HeLa/MEK2CGFP cells were transfected with p14 or NT siRNA. Starved were pretreated 75 nm LysoTracker (LT) for 30 min and then with 10 ng/mL EGF for 10 min at 37C. Images were acquired from living cells. Insets display high-magnification images of the regions of the cell indicated by white rectangles. Level pub, 10 m. To further explore the part of MP1Cp14 complex in endosomal localization of MEK2CGFP, the effect of siRNA knockdown of p14 on MEK2CGFP endosomal localization was analyzed. Because of the unavailability of the commercial antibodies to p14, the effectiveness of p14 knockdown was confirmed using cyan fluorescent protein (CFP)Cp14 transiently indicated in parental HeLa cells (Number 6B) and quantitative real-time polymerase chain reaction (RT-PCR) (Number 6C). Live cell microscopy analysis of HeLa/MEK2CGFP cells, which were preloaded with LysoTracker (to label the total population of late endosomes/lysosomes) and transfected with p14 siRNA, exposed no obvious decrease in the degree of the recruitment of MEK2CGFP to endosomes compared with cells transfected with the control nontargeting siRNA (Number 6D). At the same time, p14 depletion did not affect the general pattern of LysoTracker labeling of endosomes, although it resulted in a smaller normal size (or lower brightness) and an increased quantity of MEK2CGFP endosomes per cell, consistent with the putative part of p14 in late endosome biogenesis (28). Knockdown of p14 partially reduced the degree of EGF-induced ERK1/2 activation as exposed by western blotting and immunofluorescence staining with the phospho-ERK1/2 Rabbit polyclonal to PELI1 (pERK1/2) antibody PSI-6206 13CD3 (Number S4). Therefore, the data presented in Number 6 suggest that the MP1Cp14 complex does not look like necessary for the endosomal focusing on of MEK2CGFP, which is in agreement with the notion that MP1 is definitely a MEK1-specific scaffold (29). Clathrin-dependent endocytosis is required for MEK2 recruitment to endocytic vesicles To investigate the part of clathrin-dependent endocytosis in MEK2CGFP recruitment to endosomes, clathrin weighty chain (CHC) siRNA was depleted by siRNA. Knockdown of CHC was previously demonstrated to efficiently block the endocytosis of EGF (by 65C70%) and PSI-6206 13CD3 transferrin (by 70C80%) (30). In our experiments, the blockade of endocytosis of TfrCTR, known to internalize through coated pits, was used as an evidence of the effectiveness of CHC depletion in the individual cells. The cells were stimulated with the low concentration of EGF (2 ng/mL), conditions favoring internalization of EGFCreceptor complexes mainly through clathrin-coated pits. Live cell microscopy analysis showed that CHC siRNA PSI-6206 13CD3 PSI-6206 13CD3 dramatically abolished focusing on of MEK2CGFP to endosomes (Number 7A,B). These data shown that clathrin-dependent processes are required for MEK2CGFP recruitment to endosomes. PSI-6206 13CD3 Remarkably, the amount of phosphorylated MEK2CGFP was not modified by CHC depletion (Number 7C). Moreover, ERK1/2 activity was elevated in cells where clathrin-dependent endocytosis was inhibited (Number 7C). Therefore, clathrin-dependent endocytosis is not required for MEK and ERK activation in HeLa/MEK2CGFP cells. Open in a separate window Number 7 Effect of CHC siRNA on MEK2CGFP recruitment to endosomesA) HeLa/MEK2CGFP cells were transfected with CHC or non-targeting (NT) siRNAs and serum starved. In (A), the cells were incubated with 2 ng/mL EGF and 5 g/mL TfrCTR for 10 min at 37C and imaged through fluorescein isothiocyanate and Cy3 filter channels. Level pub, 10 m. B) Multiple images from the experiments exemplified in (A) were visually analyzed,.