Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation.

EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway.

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.