Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes" (2008).

The biological function of receptors is determined by their appropriate trafficking through the endosomal pathway. Following internalization, the transferrin (Tf) receptor quantitatively recycles to the plasma membrane, whereas the epidermal growth factor (EGF) receptor undergoes degradation. To determine how Tf and EGF engage these two different pathways we imaged their binding and early endocytic pathway in live cells using total internal reflection fluorescence microscopy (TIRF-M). We find that EGF and Tf bind to distinct plasma membrane regions and are incorporated into different endocytic vesicles. After internalization, both EGF-enriched and Tf-enriched vesicles interact with endosomes containing early endosome antigen 1 (EEA1). EGF is incorporated and retained in these endosomes, while Tf-containing vesicles rapidly dissociate and move to a juxtanuclear compartment. Endocytic vesicles carrying EGF recruit more Rab5 GTPase than those carrying Tf, which, by strengthening their association with EEA1-enriched endosomes, may provide a mechanism for the observed cargo-specific sorting. These results reveal pre-endocytic sorting of Tf and EGF, a specialized role for EEA1-enriched endosomes in EGF trafficking, and a potential mechanism for cargo-specified sorting of endocytic vesicles by these endosomes.

Treating cells with the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin causes the dissociation of the early-endosomal antigen EEA1 from early endosomes 1 . EEA1 from cytosolic extracts binds to liposomes containing phosphatidylinositol-3-phosphate (PtdIns(3)P), the major product of PI(3)K in yeast and mammalian cells 1 , 2 . Here we show that a RING zinc-finger domain at the carboxy terminus of EEA1, previously identified and named the ‘FYVE’ domain 3 , binds directly and specifically to PtdIns(3)P. This indicates that proteins containing this motif may be downstream effectors of PI(3)K in yeast and mammalian cells.

SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We performed confocal microscopy to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck was identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirmed that there was no translocation in response to growth factor and that tyrosine phosphorylation was specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains revealed differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both non-mitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm.