- Email: [email protected]
Endosomal Protein Traffic Meets Nuclear Signal Transduction Head On
Previews 161
early appearance of VRI (Cyran et al., 2003) as mechanisms to explain why per and tim mRNA levels begin to fall before PER is seen moving into the nucleus (reviewed in Hall, 2003). In terms of understanding real clocks in animals, if there is a caveat to the application of this well-crafted story, it arises only from the fact that it was necessary here to use clockless S2 cells (lacking TIM) to tease apart cause and effect. However, flies keeping time have the full armature of clock proteins: although PER is potentially capable of functioning in the early night as a repressor on its own, does it really do the heavy lifting without TIM? What are the relative contributions of PER versus PER/TIM versus VRI to the evening decline in per expression? I suspect we may not have seen the last of this discussion, but Nawathean and Rosbash have taken a major step in focusing it. And in any case, the hard trench work will now follow as the intracellular concentrations of the pertinent molecules are scrutinized, the molecular and temporal nature of the specific phosphorylations are revealed, and we come to understand how nuances in the time and place of phosphorylation can modulate the biochemistry of PER and the structural biology of its interactions at the core of the fly clock.
Endosomal Protein Traffic Meets Nuclear Signal Transduction Head On Rab5 plays a key role in controlling protein traffic through the early stages of the endocytic pathway. Previous studies on the modulators and effectors of Rab5 protein function have tied the regulation of several signal transduction pathways to the movement of protein through endocytic compartments. In the February 6, 2004, issue of Cell, Miaczynska et al. describe a surprising new link between Rab5 function and the nucleus by uncovering two new Rab5 effectors as potential regulators of the nucleosome remodeling and histone deacetylase protein complex NuRD/ MeCP1. Moving proteins through the endocytic pathway is a complex process. Proteins can enter the endocytic pathway by several routes, each of which is regulated at multiple points. Once in the cell, protein constituents then face a number of trafficking choices, including recycling back to the cell surface or traveling deeper into the endocytic pathway. Over the past 10 years, it has become increasingly evident that at many steps in the endocytic trafficking pathway endocytic cargo can interface with and influence many cellular processes (Di Fiore and De Camilli, 2001; Sorkin and Von Zastrow, 2002; von Zastrow, 2003). Differential trafficking of cell surface receptors is one way the movement of proteins through the endocytic pathway can influence cellular signal transduction processes. Once activated by ligand binding, many receptors are internalized via clathrin-coated
Jay C. Dunlap Department of Genetics Dartmouth Medical School 7400 Remsen Hanover, New Hampshire 03755 Selected Reading Ashmore, L.J., Sathyanarayanan, S., Silvestre, D.W., Emerson, M.M., Schotland, P., and Sehgal, A. (2003). J. Neurosci. 23, 7810–7819. Chang, D.C., and Reppert, S. (2003). Curr. Biol. 13, 758–762. Cyran, S.A., Buchsbaum, A.M., Reddy, K.L., Lin, M.C., Glossop, N.R., Hardin, P.E., Young, M.W., Storti, R.V., and Blau, J. (2003). Cell 112, 329–341. Darlington, T.K., Wager-Smith, K., Ceriani, M.F., Stankis, D., Gekakis, N., Steeves, T., Weitz, C.J., Takahashi, J., and Kay, S.A. (1998). Science 280, 1599–1603. Dunlap, J.C. (2003). In Chronobiology: Biological Timekeeping, J.C. Dunlap, J.J. Loros, and P. Decoursey, eds. (Sunderland, MA: Sinauer Assoc.), pp. 210–251. Hall, J.C. (2003). Adv. Genet. 48, 1–280. Nawathean, P., and Rosbash, M. (2004). Mol. Cell 13, 213–223. Rothenfluh, A., Young, M., and Saez, L. (2000). Neuron 26, 505–514. Sauman, I., and Reppert, S. (1996). Neuron 17, 889–900. Sugano, S., Andronis, C., Green, R., Wang, Z., and Tobin, E. (1998). Proc. Natl. Acad. Sci. USA 95, 11020–11025.
pits, and their journey through the endocytic pathway begins. In the case of G protein-coupled receptors (GPCRs), internalization results in one of two fates, rapid recycling to the cell surface or delivery to the lysosome and degradation. These fates are largely determined by the receptor itself and the proteins with which it associates (Spiegel, 2003; von Zastrow, 2003). Differences in these interactions and trafficking can have a strong influence on signaling by GPCRs as well as type III TGF- receptors (Chen et al., 2003). Ligand-induced clathrin-mediated endocytosis of the epidermal growth factor receptors (EGFRs) also impacts cell signaling. Attenuation of EGFR signaling can be accomplished by the internalization and degradation of the receptor (Di Fiore and De Camilli, 2001). However, like the GPCRs and the type III TGF- receptor, it has become clear that internalization of EGFR and other receptor tyrosine kinases (RTKs) does not necessarily result solely in simple downregulation of signaling cascades by the removal of activated receptors from the cell surface. It has been suggested that ligands for some RTKs, such as EGFR, may remain associated with the receptors as they travel through the early stages of the endocytic pathway, resulting in receptors that remain in their active, signaling-competent form. A number of signaling molecules can be found on endocytic structures, including Ras, Raf, and MEK, suggesting that these intracellular endocytic compartments have signaling potential (Sorkin and Von Zastrow, 2002). Rab5 is a small GTPase of the Ras superfamily that is responsible for mediating membrane trafficking events through the early stages of the endocytic pathway, and represents a key point at which endocytic trafficking can be regulated. In its GTP-bound state, Rab5 facilitates the fusion of the plasma membrane-derived endocytic vesicles with the early endosome and is also involved
Developmental Cell 162
in early endosome homotypic fusion events. Rab5 is required for constitutive as well as regulated endocytic events. Activation of Rab5 is achieved by modulating the form of guanine nucleotide bound. In the active state, Rab5 is bound to GTP, and it is inactive when bound to GDP. A number of studies have shown that activation of Rab5 (Rab5•GTP) increases flux through the early stages of the endocytic pathway. The GDP/GTP cycle of Rab5 is modulated by a number of factors that tie this cycle to cell signaling events. EGF stimulation of cells results in activation of Rin1, a Rab5 guanine nucleotide exchange factor, which in turn increases the endocytosis of the activated EGF receptor. The interaction between the EGF signaling pathway and Rab5 is also influenced by the activity of the Rab5 GTPase activating protein (GAP), RN-tre. RN-tre GAP activity, which normally deactivates Rab5 by stimulating the production of Rab5•GDP, is itself subject to negative regulation by activated EGFRs. Activation of EGFRs therefore can result in the net activation of Rab5 and an increase in EGFR internalization and EGF signal attenuation. GPCRs, RTKs, and type III TGF- receptors all interface with components of endocytic machinery to influence the signaling capacity of these receptors, primarily through modulating receptor trafficking events. A surprising new interaction between signal transduction and the endocytic pathway is described by Miaczynska et al. (2004) in the February 6 issue of Cell. They describe a novel pathway that utilizes two new Rab5 effectors to propagate signals from early endosomal structures directly to the nucleus. These effectors APPL1 (Adaptor protein containing Ph domain, PTB domain, and Leucine zipper motif) and APPL2 were identified as interaction partners of an activated version of Rab5 (Rab5•GTP␥S). APPL1 was originally identified through two-hybrid screens both as an interaction partner of AKT2, postulated to tether AKT2 to PI3K p110␣ (Mitsuuchi, Johnson et al., 1999), as well as an interaction partner of the candidate tumor suppressor DCC (Deleted in Colorectal Cancer), speculated to modulate a DCC-induced apoptotic pathway (Liu et al., 2002). Miaczynska et al. now demonstrate that both of these APPL proteins associate with cytoplasmic Rab5-positive punctate structures that are largely distributed under the plasma membrane, but are distinct from EEA1 staining endosomes. These structures appear to be unique endosomes through which EGFRs travel but not transferrin receptors. Surprisingly, activation of the EGFR leads to a drastic redistribution of APPL1, which quickly translocates from these endosomal structures to the nuclear envelope and finally on to the nucleus. The Zerial group then presents evidence indicating that APPL1 association with these endosomal structures is dependent on the presence of active, GTP-bound Rab5 and the release of APPL1
correlates with Rab5 GTP hydrolysis. These results and others indicate that Rab5 is playing a novel role in APPL1 localization by participating in the cytoplasmic sequestration of this potential signaling protein. But what is the nuclear function of these APPL proteins? To address this question, Miaczynska et al. used APPL1 coimmunoprecipitation studies to uncover APPL1 interaction partners and identified a number of components of the nucleosome remodeling and histone deacetylase NuRD/MeCP1 complex. These interactions suggested that APPL proteins may play a role in modulating cell proliferation. This model was supported by the finding that reducing APPL1 and/or APPL2 protein levels using RNA interference caused a significant reduction in the number of cells that entered S phase, suggesting that the APPL proteins do in fact play an important role in regulating cell cycle progression. These data establish a new signaling cascade that couples the Rab5 guanine nucleotide binding cycle and the regulation of cell surface receptor traffic with the spatial regulation of a cell proliferation modulator. The segregation of this APPL-signaling portion of Rab5 on specialized endosomal structures distinct from the Rab5 pool associated with constitutive endocytosis may allow the physical separation required for the independent regulation of a Rab GTPase cycle associated with regulating APPL function in response to growth factor stimulation. The identification of this new EGFR-Rab5-APPL pathway defines an intriguing new paradigm for Rab5 protein function as a participant in signal transduction systems distinct from its role in vesicular trafficking. Bruce Horazdovsky Department of Biochemistry and Molecular Biology Mayo Clinic College of Medicine Rochester, Minnesota 55905 Selected Reading Chen, W., Kirkbride, K.C., How T., Nelson, C.D., Mo, J., Frederick, J.P., Wang, X.F., Lefkowitz, R.J., and Blobe, G.C. (2003). Science 301, 1394–1397. Di Fiore, P.P., and De Camilli, P. (2001). Cell 106, 1–4. Liu, J., Yao, F., Wu, R., Morgan, M., Thorburn, A., Finley, R.L., Jr, and Chen, Y.Q. (2002). J. Biol. Chem. 277, 26281–26285. Mitsuuchi, Y., Johnson, S.W., Sonoda, G., Tanno, S., Golemis, E.A., and Testa, J.R. (1999). Oncogene 18, 4891–4898. Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler-Joseph, S., Habermann, B., Wilm, M., Parton, R.G., and Zerial, M. (2004). Cell 116, in press. Sorkin, A., and Von Zastrow, M. (2002). Nat. Rev. Mol. Cell Biol. 3, 600–614. Spiegel, A. (2003). Science 301, 1338–1339. von Zastrow, M. (2003). Life Sci. 74, 217–224.
early appearance of VRI (Cyran et al., 2003) as mechanisms to explain why per and tim mRNA levels begin to fall before PER is seen moving into the nucleus (reviewed in Hall, 2003). In terms of understanding real clocks in animals, if there is a caveat to the application of this well-crafted story, it arises only from the fact that it was necessary here to use clockless S2 cells (lacking TIM) to tease apart cause and effect. However, flies keeping time have the full armature of clock proteins: although PER is potentially capable of functioning in the early night as a repressor on its own, does it really do the heavy lifting without TIM? What are the relative contributions of PER versus PER/TIM versus VRI to the evening decline in per expression? I suspect we may not have seen the last of this discussion, but Nawathean and Rosbash have taken a major step in focusing it. And in any case, the hard trench work will now follow as the intracellular concentrations of the pertinent molecules are scrutinized, the molecular and temporal nature of the specific phosphorylations are revealed, and we come to understand how nuances in the time and place of phosphorylation can modulate the biochemistry of PER and the structural biology of its interactions at the core of the fly clock.
Endosomal Protein Traffic Meets Nuclear Signal Transduction Head On Rab5 plays a key role in controlling protein traffic through the early stages of the endocytic pathway. Previous studies on the modulators and effectors of Rab5 protein function have tied the regulation of several signal transduction pathways to the movement of protein through endocytic compartments. In the February 6, 2004, issue of Cell, Miaczynska et al. describe a surprising new link between Rab5 function and the nucleus by uncovering two new Rab5 effectors as potential regulators of the nucleosome remodeling and histone deacetylase protein complex NuRD/ MeCP1. Moving proteins through the endocytic pathway is a complex process. Proteins can enter the endocytic pathway by several routes, each of which is regulated at multiple points. Once in the cell, protein constituents then face a number of trafficking choices, including recycling back to the cell surface or traveling deeper into the endocytic pathway. Over the past 10 years, it has become increasingly evident that at many steps in the endocytic trafficking pathway endocytic cargo can interface with and influence many cellular processes (Di Fiore and De Camilli, 2001; Sorkin and Von Zastrow, 2002; von Zastrow, 2003). Differential trafficking of cell surface receptors is one way the movement of proteins through the endocytic pathway can influence cellular signal transduction processes. Once activated by ligand binding, many receptors are internalized via clathrin-coated
Jay C. Dunlap Department of Genetics Dartmouth Medical School 7400 Remsen Hanover, New Hampshire 03755 Selected Reading Ashmore, L.J., Sathyanarayanan, S., Silvestre, D.W., Emerson, M.M., Schotland, P., and Sehgal, A. (2003). J. Neurosci. 23, 7810–7819. Chang, D.C., and Reppert, S. (2003). Curr. Biol. 13, 758–762. Cyran, S.A., Buchsbaum, A.M., Reddy, K.L., Lin, M.C., Glossop, N.R., Hardin, P.E., Young, M.W., Storti, R.V., and Blau, J. (2003). Cell 112, 329–341. Darlington, T.K., Wager-Smith, K., Ceriani, M.F., Stankis, D., Gekakis, N., Steeves, T., Weitz, C.J., Takahashi, J., and Kay, S.A. (1998). Science 280, 1599–1603. Dunlap, J.C. (2003). In Chronobiology: Biological Timekeeping, J.C. Dunlap, J.J. Loros, and P. Decoursey, eds. (Sunderland, MA: Sinauer Assoc.), pp. 210–251. Hall, J.C. (2003). Adv. Genet. 48, 1–280. Nawathean, P., and Rosbash, M. (2004). Mol. Cell 13, 213–223. Rothenfluh, A., Young, M., and Saez, L. (2000). Neuron 26, 505–514. Sauman, I., and Reppert, S. (1996). Neuron 17, 889–900. Sugano, S., Andronis, C., Green, R., Wang, Z., and Tobin, E. (1998). Proc. Natl. Acad. Sci. USA 95, 11020–11025.
pits, and their journey through the endocytic pathway begins. In the case of G protein-coupled receptors (GPCRs), internalization results in one of two fates, rapid recycling to the cell surface or delivery to the lysosome and degradation. These fates are largely determined by the receptor itself and the proteins with which it associates (Spiegel, 2003; von Zastrow, 2003). Differences in these interactions and trafficking can have a strong influence on signaling by GPCRs as well as type III TGF- receptors (Chen et al., 2003). Ligand-induced clathrin-mediated endocytosis of the epidermal growth factor receptors (EGFRs) also impacts cell signaling. Attenuation of EGFR signaling can be accomplished by the internalization and degradation of the receptor (Di Fiore and De Camilli, 2001). However, like the GPCRs and the type III TGF- receptor, it has become clear that internalization of EGFR and other receptor tyrosine kinases (RTKs) does not necessarily result solely in simple downregulation of signaling cascades by the removal of activated receptors from the cell surface. It has been suggested that ligands for some RTKs, such as EGFR, may remain associated with the receptors as they travel through the early stages of the endocytic pathway, resulting in receptors that remain in their active, signaling-competent form. A number of signaling molecules can be found on endocytic structures, including Ras, Raf, and MEK, suggesting that these intracellular endocytic compartments have signaling potential (Sorkin and Von Zastrow, 2002). Rab5 is a small GTPase of the Ras superfamily that is responsible for mediating membrane trafficking events through the early stages of the endocytic pathway, and represents a key point at which endocytic trafficking can be regulated. In its GTP-bound state, Rab5 facilitates the fusion of the plasma membrane-derived endocytic vesicles with the early endosome and is also involved
Developmental Cell 162
in early endosome homotypic fusion events. Rab5 is required for constitutive as well as regulated endocytic events. Activation of Rab5 is achieved by modulating the form of guanine nucleotide bound. In the active state, Rab5 is bound to GTP, and it is inactive when bound to GDP. A number of studies have shown that activation of Rab5 (Rab5•GTP) increases flux through the early stages of the endocytic pathway. The GDP/GTP cycle of Rab5 is modulated by a number of factors that tie this cycle to cell signaling events. EGF stimulation of cells results in activation of Rin1, a Rab5 guanine nucleotide exchange factor, which in turn increases the endocytosis of the activated EGF receptor. The interaction between the EGF signaling pathway and Rab5 is also influenced by the activity of the Rab5 GTPase activating protein (GAP), RN-tre. RN-tre GAP activity, which normally deactivates Rab5 by stimulating the production of Rab5•GDP, is itself subject to negative regulation by activated EGFRs. Activation of EGFRs therefore can result in the net activation of Rab5 and an increase in EGFR internalization and EGF signal attenuation. GPCRs, RTKs, and type III TGF- receptors all interface with components of endocytic machinery to influence the signaling capacity of these receptors, primarily through modulating receptor trafficking events. A surprising new interaction between signal transduction and the endocytic pathway is described by Miaczynska et al. (2004) in the February 6 issue of Cell. They describe a novel pathway that utilizes two new Rab5 effectors to propagate signals from early endosomal structures directly to the nucleus. These effectors APPL1 (Adaptor protein containing Ph domain, PTB domain, and Leucine zipper motif) and APPL2 were identified as interaction partners of an activated version of Rab5 (Rab5•GTP␥S). APPL1 was originally identified through two-hybrid screens both as an interaction partner of AKT2, postulated to tether AKT2 to PI3K p110␣ (Mitsuuchi, Johnson et al., 1999), as well as an interaction partner of the candidate tumor suppressor DCC (Deleted in Colorectal Cancer), speculated to modulate a DCC-induced apoptotic pathway (Liu et al., 2002). Miaczynska et al. now demonstrate that both of these APPL proteins associate with cytoplasmic Rab5-positive punctate structures that are largely distributed under the plasma membrane, but are distinct from EEA1 staining endosomes. These structures appear to be unique endosomes through which EGFRs travel but not transferrin receptors. Surprisingly, activation of the EGFR leads to a drastic redistribution of APPL1, which quickly translocates from these endosomal structures to the nuclear envelope and finally on to the nucleus. The Zerial group then presents evidence indicating that APPL1 association with these endosomal structures is dependent on the presence of active, GTP-bound Rab5 and the release of APPL1
correlates with Rab5 GTP hydrolysis. These results and others indicate that Rab5 is playing a novel role in APPL1 localization by participating in the cytoplasmic sequestration of this potential signaling protein. But what is the nuclear function of these APPL proteins? To address this question, Miaczynska et al. used APPL1 coimmunoprecipitation studies to uncover APPL1 interaction partners and identified a number of components of the nucleosome remodeling and histone deacetylase NuRD/MeCP1 complex. These interactions suggested that APPL proteins may play a role in modulating cell proliferation. This model was supported by the finding that reducing APPL1 and/or APPL2 protein levels using RNA interference caused a significant reduction in the number of cells that entered S phase, suggesting that the APPL proteins do in fact play an important role in regulating cell cycle progression. These data establish a new signaling cascade that couples the Rab5 guanine nucleotide binding cycle and the regulation of cell surface receptor traffic with the spatial regulation of a cell proliferation modulator. The segregation of this APPL-signaling portion of Rab5 on specialized endosomal structures distinct from the Rab5 pool associated with constitutive endocytosis may allow the physical separation required for the independent regulation of a Rab GTPase cycle associated with regulating APPL function in response to growth factor stimulation. The identification of this new EGFR-Rab5-APPL pathway defines an intriguing new paradigm for Rab5 protein function as a participant in signal transduction systems distinct from its role in vesicular trafficking. Bruce Horazdovsky Department of Biochemistry and Molecular Biology Mayo Clinic College of Medicine Rochester, Minnesota 55905 Selected Reading Chen, W., Kirkbride, K.C., How T., Nelson, C.D., Mo, J., Frederick, J.P., Wang, X.F., Lefkowitz, R.J., and Blobe, G.C. (2003). Science 301, 1394–1397. Di Fiore, P.P., and De Camilli, P. (2001). Cell 106, 1–4. Liu, J., Yao, F., Wu, R., Morgan, M., Thorburn, A., Finley, R.L., Jr, and Chen, Y.Q. (2002). J. Biol. Chem. 277, 26281–26285. Mitsuuchi, Y., Johnson, S.W., Sonoda, G., Tanno, S., Golemis, E.A., and Testa, J.R. (1999). Oncogene 18, 4891–4898. Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler-Joseph, S., Habermann, B., Wilm, M., Parton, R.G., and Zerial, M. (2004). Cell 116, in press. Sorkin, A., and Von Zastrow, M. (2002). Nat. Rev. Mol. Cell Biol. 3, 600–614. Spiegel, A. (2003). Science 301, 1338–1339. von Zastrow, M. (2003). Life Sci. 74, 217–224.