Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Ret regulates phosphorylation and protein levels of Pcdhα and Pcdhγ. (A) Differentiated CAD cells expressing Pcdhα4-TAP (α4-TAP), Pcdhγb7-TAP (γb7-TAP), or empty TAP vector (TAP) were left untreated or stimulated with GDNF/GFRα1 for 0.5 or 3 h. Anti-FLAG (TAP) immunoprecipitates (IPs) were blotted with anti-phosphotyrosine (anti-P-Tyr). The blot was reprobed with an anti-HA (TAP) antibody. Total cell lysates were blotted with anti-MAPK. (B) CAD cells expressing Pcdhα4-ΔC3-TAP (ΔC3-TAP), Pcdhα4-TAP, Pcdhγb7-TAP, or empty TAP vector were infected with anti-Ret shRNA or control anti-GFP shRNA lentivirus. Anti-FLAG (TAP) IPs of differentiated cells were blotted for P-Tyr, and the blot was reprobed with an anti-HA (TAP) antibody. Total lysate was blotted with an anti-Ret antibody. (C) Total lysate of differentiated CAD cells expressing lentiviral shRNA plasmids targeting Ret or GFP was blotted for Ret9 and Ret51, Pcdhα, Pcdhγ, and TuJ1.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Phospho-proteomics, Expressing, Plasmid Preparation, Infection, shRNA, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Pcdhγ undergoes GDNF-induced phosphorylation and interacts with Ret in MNs and sympathetic neurons. (A) Anti-Pcdhγ or control rabbit serum (IgG) immunoprecipitates (IPs) of MN lysate were blotted with anti-Ret and anti-P-Tyr antibodies. The blot was reprobed with an anti-Pcdhγ antibody. (B) MNs were left untreated, stimulated for 30 min with GDNF/GFRα1, or treated for 20 min with PP2 prior to 30 min of GDNF/GFRα1 stimulation. Anti-Pcdhγ or rabbit serum control (IgG) IPs were blotted with anti-P-Tyr and anti-Pcdhγ. Total cell lysate was blotted with anti-Ret, anti-P-MAPK, and anti-MAPK. (C) Sympathetic neurons were left untreated or stimulated for 30 min with GDNF. Anti-Pcdhα and anti-Pcdhγ IPs were blotted with anti-P-Tyr and anti-pan-Ret. The blots were reprobed with anti-Pcdhα and anti-Pcdhγ antibody. Total cell lysate was blotted with anti-Ret and anti-Tuj1. (D) Cortical glia were left untreated, stimulated with GDNF/GFRα1 for 30 min, or treated with PP2 before 30 min of GDNF/GFRα1 stimulation. Anti-Pcdhγ or rabbit control serum (IgG) IPs were blotted with anti-P-Tyr antibody. The blot was reprobed with an anti-Pcdhγ antibody.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Phospho-proteomics, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Pcdhs are required for stabilization of activated Ret. (A) Differentiated CAD cells stably expressing Pcdhα4-TAP (α4-TAP), Pcdhγb7-TAP (γb7-TAP), or TAP vector control (TAP) were left untreated or treated with GDNF/GFRα1 for 0.5 or 3 h. Anti-Ret51 and anti-FLAG (TAP) immunoprecipitates (IPs) were blotted with anti-Ret51 and anti-P-Ret. The blots were reprobed with anti-Ret51 antibody. The anti-FLAG (TAP) immunoprecipitation blot was reprobed with anti-HA antibody. (B) CAD cells infected with anti-Pcdhα, anti-Pcdhγ, anti-GFP, or anti-Ret lentiviral shRNA were differentiated. Total cell lysate was blotted for Ret9, Ret51, Pcdhα, Pcdhγ, and Tuj1. (C) Sympathetic neurons were infected with lentivirus encoding shRNA plasmids targeting Pcdhα and Pcdhγ or GFP. Neurons were cultured for an additional 7 d and stimulated with GDNF for various times. Anti-Ret51 IPs were blotted with anti-Ret51 and anti-P-Ret. Antiubiquitin IPs were blotted with anti-Ret51. Total cell lysate was blotted with anti-P-Ret, anti-Ret51, Pcdhα, Pcdhγ, and Tuj1 antibodies.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Infection, shRNA, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Model of Pcdh and Ret interaction and stabilization in CAD and sympathetic neurons. Activation of Ret with GDNF/GFRα1 leads to ubiquitination and rapid degradation. Pcdhs not bound to Ret also undergo degradation. Activated Ret bound to Pcdh is stabilized. Pcdhs bound to activated Ret are stabilized and phosphorylated and might initiate downstream signaling.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Activation Assay, Ubiquitin Proteomics

Journal: JNCI Journal of the National Cancer Institute

Article Title: Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves

doi: 10.1093/jnci/djp456

Figure Lengend Snippet: Glial cell-derived neurotrophic factor–induced cancer cell invasion. A) Serum-starved MiaPaCa2 cells were plated in the upper chamber, and GDNF (1–100 ng/mL) was added to the lower chamber of transwell plates and cultured for 24 hours (white bars). In another experiment, dorsal root ganglion (DRG) nerve cells were grown on the lower plate instead of adding GDNF (the condition is indicated below the graph). While nerves induced cancer cell migration (control—blue bar), adding anti-GDNF antibodies to the lower chamber suppressed it (red bars). Nonimmune Igs were used for control (n = 10). Yellow bars: lentiviral gene transduction of neurons with short hairpin RNA (shRNA) targeting GDNF expression (shGDNF) also inhibited cancer cell migration toward the neurons (n = 6). An empty viral vector served as the control (shControl). B) Immunoblots of conditioned media from dissociated DRG nerve cell cultures. Conditioned media was recovered at 24–48 hours after incubation. Protein analysis showed detection of a protein band, which corresponds to the secreted form of GDNF. C) Immunoblots of GDNF protein recovered from nerve cell lysates after lentiviral gene transduction with shRNA directed against GDNF (n = 3–6 experiments in each condition). Empty vector lentivirus was used as control (shControl). D) Cancer cells (green asterisk) were grown in Matrigel adjacent to DRG (white asterisk). Pictures show representative experiments of neural invasion by MiaPaCa2 cells taken at day 10 in control conditions allowing for neural invasion (left); with anti-GDNF antibodies inhibiting invasion (middle) and of a DRG from a heterozygous mouse deficient of GDNF (gdnf−/+). The black arrows indicate the invading cancer cells. E) An illustration showing the calculation of nerve invasion index (α/β). F) Dose–response curves showing the effect of anti-GDNF antibodies on invasion index at day 10 (P < .001; n = 5–15 experiments in each condition). Nonimmune Igs served as control (open circle). G) Nerve invasion index in wild-type (WT; white bar) and gdnf−/+ mice (gray bar) at day 10 (n = 60–66). P values in (A, F, and G) were calculated by two-sided Student t test. DMEM = Dulbecco's modified Eagle medium; hpf = high-power field.

Article Snippet: Lentiviral Gene Transduction and RNA Interference for Inhibition of GDNF Expression by DRG Lentiviral pLKO.1 vectors expressing short hairpin RNA against murine GDNF ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010275","term_id":"672349279"}} NM_010275 ) were purchased from Open Biosystems (Huntsville, AL) and from OriGene Technologies (Rockville, MD). pLKO.1 empty vector was used as a negative control.

Techniques: Derivative Assay, Cell Culture, Migration, Control, Transduction, shRNA, Expressing, Plasmid Preparation, Western Blot, Incubation, Modification

Journal: JNCI Journal of the National Cancer Institute

Article Title: Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves

doi: 10.1093/jnci/djp456

Figure Lengend Snippet: Activation of RET and mitogen-activated protein kinase in neuroinvasive cancer cells. A) Expression of RET (170/150 kDa) and GFRα1 (53 kDa) in various cancer cell lines, as determined by immunoblotting. Expression of β-actin (42 kDa) was used as a control for loading and transfer. B) Cancer cell colonies (green asterisk) were grown in Matrigel adjacent to dorsal root ganglion (DRG) (white asterisk). When the DRG neurites made contact with the cancer colony, anti-RET antibodies (1 μg/mL) or recombinant mouse RET-Fc chimera (5 μg/mL) were added to the media. Pictures show representative experiments of neural invasion by MiaPaCa2 cells taken at day 10. Nonimmune antibodies were used as control (n = 8–12). C) The effect of small interfering RNA (siRNA) directed against RET (siRET) on invasion of pancreatic cancer cells along DRG nerves. siRET reduced RET expression within 72 hours after transfection (immunoblotting—lower panel), whereas noncoding siRNA served as control (siControl) and had no impact on RET expression. Nerve invasion was measured in vitro on day 7 (n = 11–17). D) Dose–response effect of the RET inhibitor pyrazolopyrimidine-1 (PYP1) on MiaPaCa2 cell invasion index. Inset: effect of PYP1 on cell migration toward DRG nerve cells in dual chamber migration assays (high-power field [hpf], n = 10). E) Dose–response curves of the effect of the MEK-1 inhibitor PD98059 on MiaPaCa2 nerve invasion index (P < .001; n = 5–15 experiments in each condition). F) Effect of PYP1 and PD98059 on invasion index of another human pancreatic adenocarcinoma cell line—Panc1, and on a human cervical cancer cell line—HeLa (*P < .001; n = 10 experiments in each condition). G) Cell viability was measured after treatment with PD98059 (25 μM) and PYP1 (2 μM) (n = 3 in each condition). H) Immunoblotting to assay GDNF expression by the DRG after treatment with PD98059 and PYP1. P values were calculated by using a two-sided Student t test.

Article Snippet: Lentiviral Gene Transduction and RNA Interference for Inhibition of GDNF Expression by DRG Lentiviral pLKO.1 vectors expressing short hairpin RNA against murine GDNF ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010275","term_id":"672349279"}} NM_010275 ) were purchased from Open Biosystems (Huntsville, AL) and from OriGene Technologies (Rockville, MD). pLKO.1 empty vector was used as a negative control.

Techniques: Activation Assay, Expressing, Western Blot, Control, Recombinant, Small Interfering RNA, Transfection, In Vitro, Migration

Journal: JNCI Journal of the National Cancer Institute

Article Title: Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves

doi: 10.1093/jnci/djp456

Figure Lengend Snippet: Activation of RET and mitogen-activated protein kinase in neuroinvasive cancer cells. A) Time-lapse image analysis of cells contacting a nerve, without nerve contact, with contact in the presence of PD98059 (25 μM) or with contact in the presence of pyrazolopyrimidine-1 (PYP1) (2 μM). Bars show the mean distance of cells migrated from the point of origin and the mean velocity of cancer cell migration (two-sided analysis of variance with multiple range test adjusted for contrasts with control, P < .001; n = 8–21 in each group). B) Graphs representing migration of cancer cells along nerves with or without the MEK-1 inhibitor PD98059 (25 μM) or the RET inhibitor PYP1 (2 μM). The distance from origin is shown for each cell (n = 8–21), see Figure 2, C for control. C) MiaPaCa2 cells were conditioned with recombinant GDNF (100 ng/mL) for 0–40 minutes at room temperature. Incubation with GDNF induced phosphorylation of extracellular signal–regulated kinase (ERK) as measured by enzyme-linked immunosorbent assay. The relative expression of P-ERK and ERK at various time points after stimulation is shown. Comparisons were between absorption read at time point 0 minutes (control) and 40 minutes (poststimulation). (Relative expression of P-ERK 1.07 vs 1.45, for time point 0 and 40 minutes, respectively, difference = 0.38, 95% confidence interval on the means = 0.91 to 1.23 vs 1.30 to 1.60, respectively, n = 6 for each point, *P < .001.) D) MiaPaCa2 and QLL2 cells were grown in Matrigel alone or with dorsal root ganglion (DRG). Following serum starvation, cancer cells were microscopically separated from the DRG and total cancer cells lysates were immunoblotted with anti P-ERK and ERK antibodies. E) PYP1 (2 μM) treatment on ERK phosphorylation in MiaPaCa2 cells cultured with DRG. F) Top: incubation of MiaPaCa2 cells with GDNF (100 ng/mL) and phosphorylation of RET (P-RET) within 20–40 minutes. Bottom: dissociated nerves were grown in culture with serum-free Dulbecco's modified Eagle medium, and the conditioned media was collected after 48 hours and added to serum-starved MiaPaCa2 cells. Immunoblotting was used to detect P-RET within 10–40 minutes after adding the conditioned media to the cells. P values were calculated by using a two-sided Student t test.

Article Snippet: Lentiviral Gene Transduction and RNA Interference for Inhibition of GDNF Expression by DRG Lentiviral pLKO.1 vectors expressing short hairpin RNA against murine GDNF ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010275","term_id":"672349279"}} NM_010275 ) were purchased from Open Biosystems (Huntsville, AL) and from OriGene Technologies (Rockville, MD). pLKO.1 empty vector was used as a negative control.

Techniques: Activation Assay, Migration, Control, Recombinant, Incubation, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Modification, Western Blot