Transport routes of nucelar receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) generally represent integral transmembrane proteins with one membrane spanning domain, which evoke intracellular signaling cascades after extracellular binding of polypeptide growth factors. Several RTKs, such as EGFR and FGFR-1, also localize to the cell nucleus; however, both functions and transport pathways leading to nuclear localization of RTKs are presently poorly understood. This project therefore aims at elucidating the cellular transport pathways of nuclear RTKs, using laser scanning confocal microscopy and biochemical approaches such as cell fractionation. Available evidence suggests that nuclear RTKs exist in a non-membrane bound form and are imported into the nucleus from the cytoplasm by well- described pathways such as the importin beta pathway. This opens one key question of the proposal: Are transmembrane proteins able to leave the membranous fraction of the cell and how is this membrane extraction achieved? The ER associated degradation (ERAD) system, a multi-component quality control system is able to retrotranslocate luminal and transmembrane ER proteins to the cytosol for degradation. A number of bacterial toxins and viruses are known to avoid subsequent degradation and accumulate in the cytosol after retrotranslocation. This indicates that RTKs could be extracted from the membrane fraction at the endoplasmic reticulum (ER) by the ERAD system. This would require, however, that RTKs determined for nuclear import are first transported to the ER, which opens another key question of the project: Are plasma-membrane bound RTKs transported to the nucleus in response ligand stimulation or are nuclear RTKs imported at an early step after protein synthesis, thus originating rather from intracellular sources (ER)? If nuclear RTKs are originating from the plasma membrane, it is very likely that nuclear transport is dependent on ligand-induced endocytosis, and retrograde transport the influence of which is going to be addressed. Technologically, the project aims addressing the relevant biological questions using photoactivateable GFP and functional live cell microscopy in combination with cell fractionation techniques. Summarizing current evidence suggests that RTKs are transported into the nucleus from either the plasma membrane (by endocytosis) or intracellular sites in a multi-step pathway and are finally extracted from the membrane fraction by the ERAD system into the cytosol, from where nuclear import finally occurs. Summarizing, the project is expected to substantially increase the current understanding of the transport routes leading to nuclear localization of RTKs, which is of potential translational interest, since the nuclear RTK localization has very recently been found to be associated with poor prognosis in human cancers.

Receptor tyrosine kinases (RTKs) generally represent integral transmembrane proteins with one membrane spanning domain, which evoke intracellular signaling cascades after extracellular binding of polypeptide growth factors. Several RTKs, such as EGFR and FGFR-1, also localize to the cell nucleus; however, both functions and transport pathways leading to nuclear localization of RTKs are presently poorly understood. This project therefore aims at elucidating the cellular transport pathways of nuclear RTKs, using laser scanning confocal microscopy and biochemical approaches such as cell fractionation. Available evidence suggests that nuclear RTKs exist in a non-membrane bound form and are imported into the nucleus from the cytoplasm by well- described pathways such as the importin beta pathway. This opens one key question of the proposal: Are transmembrane proteins able to leave the membranous fraction of the cell and how is this membrane extraction achieved? The ER associated degradation (ERAD) system, a multi-component quality control system is able to retrotranslocate luminal and transmembrane ER proteins to the cytosol for degradation. A number of bacterial toxins and viruses are known to avoid subsequent degradation and accumulate in the cytosol after retrotranslocation. This indicates that RTKs could be extracted from the membrane fraction at the endoplasmic reticulum (ER) by the ERAD system. This would require, however, that RTKs determined for nuclear import are first transported to the ER, which opens another key question of the project: Are plasma-membrane bound RTKs transported to the nucleus in response ligand stimulation or are nuclear RTKs imported at an early step after protein synthesis, thus originating rather from intracellular sources (ER)? If nuclear RTKs are originating from the plasma membrane, it is very likely that nuclear transport is dependent on ligand-induced endocytosis, and retrograde transport the influence of which is going to be addressed. Technologically, the project aims addressing the relevant biological questions using photoactivateable GFP and functional live cell microscopy in combination with cell fractionation techniques. Summarizing current evidence suggests that RTKs are transported into the nucleus from either the plasma membrane (by endocytosis) or intracellular sites in a multi-step pathway and are finally extracted from the membrane fraction by the ERAD system into the cytosol, from where nuclear import finally occurs. Summarizing, the project is expected to substantially increase the current understanding of the transport routes leading to nuclear localization of RTKs, which is of potential translational interest, since the nuclear RTK localization has very recently been found to be associated with poor prognosis in human cancers.