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First published online 29 November 2005
doi: 10.1242/jcs.02695

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Role of the subcellular localization of ALK tyrosine kinase domain in neuronal differentiation of PC12 cells

INSERM, U706, Institut du Fer à Moulin, 17 rue du Fer à Moulin; and UPMC, 4 Place Jussein, Paris, F-75005, France

View larger version (24K): [in a new window]   Fig. 1. ALK-FH-derived proteins and the inducible dimerization system. (A) Schematic representation of the induced dimerization of ALK-FH protein by the synthetic lipophylic and bifunctional dimerizer AP20187. (B) Schematic representation of the full-length ALK-FH protein and its intracellular domain as membrane-bound (transmembrane, tmbIA-FH; or myristoylated, myrIA-FH) or cytosolic (cytoIA-FH) proteins. ECD, extracellular domain; TM, transmembrane domain; PTK, protein tyrosine kinase domain; 2x FKBPv, tandem copies of modified form of the intracellular FK506-binding protein; HA, hemagglutinin tag; myr, myristoylation sequence from Src.

View larger version (38K): [in a new window]   Fig. 2. Induced kinase activation of the full-length receptor ALK results in neurite extension and ERK 1/2 activation. (A) Dimerizer dose-dependent effect on neurite outgrowth induced by the activation of ALK-FH protein. PC12 cells were electroporated with the ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated without or with increasing concentrations of dimerizer as indicated for 2 days. Then immunofluorescence assay was performed using the anti-HA-tag antibody (12CA5); 100 transfected cells were counted and cells bearing neurites longer than twice the diameter of the cell body were scored as differentiated. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (B) Neurite outgrowth induced by ALK-FH dimerization is a result of its own kinase activity. PC12 cells were electroporated with the indicated constructs (25 µg) and then submitted to the same protocol as described in (A). Cells were treated with a dimerizer concentration of 20 nM. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Time course of (C) ALK-FH and (D) ERK 1/2 phosphorylation following dimerizer treatment. ALK-FH-transfected PC12 cells were incubated with the dimerizer (20 nM) for the indicated periods. As controls, non-transfected cells (Mock) or cells transfected with the dALK-FH mutant were treated with the dimerizer during 30 minutes. Cells were lysed in a RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-PY (4G10) or the anti-P-ERK 1/2 antibody (upper panels) and then reprobed with the indicated antibodies (lower panels). The P-ERK 2 densitometry quantification was performed using the Odyssey Imaging System on three independent experiments. Statistical analyses were carried out by Student's t-test (***P<0.005). Asterisk in C indicates a 160 kDa protein that was not revealed by the anti-HA-tag antibody and therefore not related to ALK.

View larger version (39K): [in a new window]   Fig. 3. Cellular expression of ALK-FH-derived proteins: normalization of the expression levels and subcellular localization. (A) Western blot analysis of the expression of ALK-FH-derived proteins after normalization of their expression levels. PC12 cells were electroporated with the indicated quantities of the DNA constructs, in order to obtain comparable levels of protein expression. Cells were lysed after two days in a RIPA buffer as described in the Materials and Methods, and the lysates (10 µg) were submitted to a western blot analysis using the anti-HA-tag (12CA5) monoclonal antibody. (B) Immunofluorescence confocal microscopy analysis of the subcellular localization of ALK-FH-derived proteins revealed with the anti-HA-tag antibody (green) in permeabilized PC12 cells (representative fields are shown). Cell nuclei were labeled with the Sytox Orange nucleic acid stain (red). Bar, 10 µm.

View larger version (26K): [in a new window]   Fig. 4. Membrane attachment of the ALK intracellular domain is required to induce neurite extension of PC12 cells and MAP kinase activation. (A) PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for 2 days. Then cells were fixed, permeabilized and immunofluorescence assay was performed. Cells expressing the ALK-FH-derived proteins were labeled with the anti-HA-tag antibody (12CA5) and cell nuclei were colored with the nucleic acid stain Hoechst 33258. Bar, 100 µm. (B) Quantification of the effect of the induced activation of the different proteins on neurite outgrowth, as indicated in the Materials and Methods. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Time course of ERK 1/2 phosphorylation following dimerizer treatment. PC12 cells expressing the indicated proteins were cultured overnight in a 1% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-HA-tag or the anti-P-ERK antibody and then reprobed with the anti-PY (4G10) or the anti-ERK antibody.

View larger version (19K): [in a new window]   Fig. 5. Activation of the MAP kinase pathway, but not the PI 3-kinase pathway, is required to induce neurite extension of PC12 cells. (A) MEK inhibitor dose-dependent inhibition of neurite outgrowth induced by the activation of the full-length ALK-FH protein. PC12 cells were electroporated with ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for two days in the presence of increasing concentrations of the MEK inhibitor U0126 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO control (0 µM) showed no adverse effects. (B,C) Inhibition of neurite outgrowth by the MEK inhibitor U0126 but not by the PI 3-kinase inhibitor wortmannin. PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured as described above and treated with the dimerizer (20 nM) in the presence or not of U0126 (25 µM) (B) or wortmannin (50 nM) (C) for two days. All the data (A-C) were obtained following immunofluorescence detection with the anti-HA-tag antibody (12CA5), neurite outgrowth of transfected cells being scored as indicated in the Materials and Methods. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%).

View larger version (13K): [in a new window]   Fig. 6. Neurite outgrowth of PC12 cells mediated by ALK-FH-derived protein is accompanied by arrest of DNA synthesis. Cells were electroporated with normalized quantities of the indicated constructs, continually cultured in a normal serum medium (10% horse serum, 5% fetal calf serum) in order to study their normal asynchronous growth and treated with the dimerizer (20 nM) for 12 hours in the presence or not of U0126 (25 µM). At 6 hours after dimerizer application, cells were incubated with BrdU (10 µM) for 6 hours of incorporation. Then immunofluorescence assay was performed using the anti-HA-tag (3F10) and the FITC-conjugated anti-BrdU antibodies; 100 transfected cells were counted and cells with positive BrdU staining were scored as having undergone DNA replication during the time of labeling. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Statistical analyses were carried out by Student's t-test (*P<0.05; **P<0.01).

View larger version (34K): [in a new window]   Fig. 7. Activation of the cytosolic form of the ALK intracellular domain induces DNA synthesis through the PI 3-kinase/AKT pathway. (A) Induced activation of the cytoIA-FH but not of the myrIA-FH protein leads to DNA synthesis in PC12 cells. Cells were electroporated with normalized quantities of the indicated constructs and were submitted to the same protocol as in Fig. 6 except that they were cultured overnight in a 1% horse serum medium before dimerizer treatment. ***P<0.005 (Student's t-test). (B) PI 3-kinase inhibitor inhibition of DNA synthesis induced by the activation of the cytoIA-FH protein. PC12 cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. They were treated with the dimerizer (20 nM) for 12 hours in the presence of the indicated increasing concentrations of PI 3-kinase inhibitors wortmannin or LY294002 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO controls (0 nM or µM) showed no adverse effects. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Induced DNA synthesis of PC12 cells expressing the cytoIA-FH protein is dependent on PI 3-kinase but not on ERK 1/2. Cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. U0126 (25 µM) and/or wortmannin (50 nM) were added to the medium 60 and 30 minutes, respectively, before dimerizer treatment. **P<0.01; ***P<0.005 (Student's t-test). (D) Time course of AKT phosphorylation following dimerizer treatment. PC12 cells transfected with the indicated constructs were cultured overnight in 0% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-AKT phosphoserine-473 antibody and then reprobed with the anti-AKT antibody. (E) MAP kinase pathway inhibition revealed a potential activation effect of myrIA-FH protein on DNA synthesis through the PI 3-kinase pathway. Cells transiently expressing the myrIA-FH protein were submitted to the same protocol as in C. **P<0.01 (Student's t-test).