The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14–MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14–MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.

Late endosomes locally regulate cell migration by transporting the p14–MP1 scaffold complex to the vicinity of focal adhesions.

Epidermal growth factor receptor (EGFR) signaling is initiated by ligand binding followed by homodimerization and rapid receptor autophosphorylation. Monitoring EGFR phosphorylation was achieved by measuring translocation and binding of an enhanced yellow fluorescent protein (EYFP)-labeled phosphotyrosine-binding domain (PTB) to enhanced cyan fluorescent protein (ECFP)-tagged EGFR using fluorescence lifetime imaging microscopy or sensitized emission measurements. To simplify dynamic phosphorylation pattern measurements in cells, FLAME, a ratiometric sensor containing both EGFR-ECFP and PTB-EYFP in one molecule, was designed and examined in COS7 cells. Epidermal growth factor (EGF) treatment demonstrated rapid and reversible changes in the EYFP/ECFP fluorescence emission ratios, due to binding of the PTB domain to its consensus binding sites upon phosphorylation at the cell periphery, whereas perinuclear regions failed to respond to EGF but were responsive to tyrosine kinase inhibition. Long-term EGF treatment resulted in accumulation of dephosphorylated receptor in the perinuclear region due to active dephosphorylation occurring at intracellular sites. This indicates that the sensor closely approaches the true dynamics of tyrosine kinase autophosphorylation and dephosphorylation. Phosphatase inhibition by pervanadate resulted in an irreversible response in all cellular compartments. These data show that EGFR is under tonic phosphatase suppression maintaining the receptor in an unphosphorylated (silent) state and is dephosphorylated at endomembranes after ligand-mediated endocytosis. The epidermal growth factor receptor (EGFR)1 tyrosine kinase is a transmembrane receptor, which exerts its activity through extracellular binding of epidermal growth factor (EGF) and EGF-like growth factors. Ligand-stimulated EGFR homoand heterodimerizes and subsequently phosphorylates itself and other cytoplasmic substrates at tyrosine residues, thus activating an intracellular signaling cascade and finally resulting in a wide range of responses, such as cytoskeletal rearrangements, changes in gene expression, and increased cell proliferation (1). Previous analysis of the phosphorylation/activation status of the EGFR using a number of biochemical approaches such as immunoprecipitation and/or immunoblotting has revealed the basic mechanisms of ligand-induced autophosphorylation. Moreover, cell-based assays using fluorescence lifetime measurements have yielded quantitative spatial information about EGFR phosphorylation in fixed cells (2). Unfortunately, biochemical as well as fixed cell approaches generally fail to capture the dynamics of tyrosine kinase activation in a single cell.Therefore, the activity of EGFR has been elegantly monitored in live cells expressing genetically encoded ECFP-and EYFP-labeled ratiometric fluorescence resonance energy transfer (FRET) sensors on downstream targets such as Ras (3) or cytoplasmic substrates (4). The latter study specifically monitored the enzymatic activity of the EGFR tyrosine kinase towar...