Fyn tyrosine kinase increases Apolipoprotein E Receptor 2 levels and phosphorylation.

Apolipoprotein E Receptor 2 (ApoER2) and the tyrosine kinase Fyn are both members of the Reelin pathway, a signaling pathway essential for the laminar formation of the cortex during development and proper dendritic spine density and long-term potential (LTP) in the adult brain. In the presence of extracellular Reelin, ApoER2 binds the intracellular protein Dab1, an adaptor protein that is phosphorylated by Fyn. However, direct interactions between ApoER2 and Fyn are not well defined. Here, we show that total levels of ApoER2 and surface levels of ApoER2 are increased by active Fyn. Via a separate mechanism, ApoER2 is also phosphorylated by Fyn, an event that peaks in the postnatal cortex at day 5 and can occur at multiple ApoER2 tyrosine residues. Dab1 is also involved in this phosphorylation, promoting the phosphorylation of ApoER2 by Fyn when it is itself phosphorylated. These results elucidate some of the intracellular mechanisms that give rise to a functional Reelin pathway

Fyn increases the interaction between ApoER2 and Dab1.

<p>COS7 cells were co-transfected with double-tagged ApoER2 and either Fyn, Dab1, or Fyn and Dab1 and lysates collected in IP buffer after 20 hours. A. Lysates were immunoprecipitated (IP) with either GFP (for Dab1) or rabbit IgG, run on a Western blot, and probed for HA (for ApoER2). Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and Tubulin. B. The lysates used in A were precipitated with either HA (for ApoER2) or mouse IgG, and analyzed for Dab1-GFP. C. Quantification of lanes 2 and 3 from A. *<i>p<0.05 (t-test). Data are mean +/− SD. N = 3.</i> D. COS7 cells were co-transfected as in A and treated with either DMSO (control) or PP2 for 6 hours. Lysates were collected and immunoprecipitated as in A.</p

ApoER2 levels increase independently of the intracellular domain of ApoER2.

<p>A. Schematic of tagged ApoER2 constructs. Wild-type (WT) murine ApoER2 with both an N-terminal HA tag and a C-terminal myc tag. ΔICD is missing amino acids 731–841 and the myc tag. SP: Signal peptide. B. COS7 cells were co-transfected with either ApoER2 WT or ΔICD and either Fyn or empty vector. Lysates were collected in RIPA buffer and analyzed for HA, Fyn, and Tubulin. *<i>p<0.05,</i> **<i>p<0.01 vs empty vector (Bonferroni’s Multiple Comparisons test). Data are mean +/− SD. N = 5.</i></p

Fyn phosphorylation of ApoER2 is increased in the presence of Dab1.

<p>A. COS7 cells were transfected with untagged ApoER2 and either empty vector, Fyn, Dab1-WT, or Fyn and Dab1-WT together. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, run on a Western blot, and probed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and β–actin. B–C. COS7 cells were transfected and analyzed as in A, but with Dab1-ab (B) or Dab1-cd (C) instead of Dab1-WT.</p

Fyn increases ApoER2 levels.

<p>A. Brains from 3 week old wild-type (WT) and <i>Fyn</i> knock-out (KO) mice were homogenized in RIPA buffer and analyzed for ApoER2, Fyn, or β-tubulin by immunoblot. ApoER2 levels were normalized to β-tubulin. *<i>p<0.05 (t-test); Data are mean +/− SD. N = 3.</i> B. COS7 cells co-transfected with Myc-tagged ApoER2 and empty vector (vec) or Fyn were collected in RIPA buffer and analyzed as above. ApoER2-Myc levels were normalized to β-actin. *<i>p<0.05 (t-test); Data are mean +/− SD. N = 3.</i> C. COS7 cells co-transfected with Myc-tagged VLDLR and empty vector or Fyn were collected in RIPA buffer, Western blotted, and probed as above. *<i>p<0.05 (t-test). Data are mean +/− SD. N = 3.</i> D. COS7 cells co-transfected with ApoER2 and empty vector or Fyn were subject to cell surface biotinylation. Lysates were collected, purified with an avidin gel and Western blotted for ApoER2. Corresponding cell lysates that were not biotinylated were collected in PBS and probed for total ApoER2, Fyn, or β-actin. Biotinylated (surface) and total ApoER2 levels were quantified. ***<i>p<0.001 (Bonferroni’s Multiple Comparison test). NS: non-significant. Data are mean +/− SD. N = 3.</i> E. COS7 cells co-transfected with ApoER2 and either empty vector or Fyn were treated with cycloheximide. Lysates were collected after 0, 15, 30, 60, 120, and 360 minutes and Western blotted for ApoER2. For graphing, levels were normalized to the 0 time point. Nonlinear regression lines (solid) are shown. ****<i>p<0.0001; the two curves are significantly different. Data are mean +/− SD. N = 3.</i></p

Fyn phosphorylates ApoER2.

<p>A. COS7 cells were co-transfected with untagged ApoER2 and Fyn or empty vector. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Lane numbers are indicated for clarity. B. Schematic of WT ApoER2 and the mutant with all three intracellular tyrosines mutated to phenylalanines (tYF). The signal peptide (SP), N-terminal HA tag, and C-terminal myc tag are indicated. C. COS7 cells were co-transfected with different ApoER2 mutants (2 single mutants on left, 1 single mutant and triple mutant on right) and Fyn or empty vector and immunoprecipitated as in A, and analyzed for myc. Below are Western blots showing total levels of ApoER2, Fyn, and β–actin. D. Quantification of the upper-phosphorylated ApoER2 band (indicated by arrow in boxed example) over total ApoER2 from C. **<i>p<0.01,</i> ***<i>p<0.001 vs empty vector (Bonferroni’s Multiple Comparisons test). Data are mean +/− SD. N = 3.</i></p

Fyn’s effects on ApoER2 levels depend on Fyn’s kinase activity.

<p>A. COS7 cells were co-transfected with ApoER2 and either Fyn or empty vector and treated with PP2 or DMSO (control) for 6 hours. Lysates were collected in RIPA buffer, Western blotted and probed for ApoER2, Fyn, and tubulin. **<i>p<0.01 compared to empty vector treatment;</i> †† <i>p<0.01 compared to control treatment with Fyn (Bonferroni’s Multiple Comparison test). NS: non-significant. Data are mean +/− SD. N = 3.</i> B. COS7 cells were co-transfected with ApoER2 and either empty vector, constitutively-active Fyn (Fyn CA) or kinase-dead Fyn (Fyn KD). Lysates were collected in RIPA buffer, Western blotted and ApoER2 levels were quantified. *<i>p<0.05 (Bonferroni’s Multiple Comparison test). Data are mean +/− SD. N = 3.</i> C. At DIV 5, primary cortical neurons were treated with PP2 for 24 hours. Lysates were collected in RIPA buffer and Western blotted for ApoER2, 4G10, and actin. Total ApoER2 levels (both upper and lower bands) were normalized to actin. *<i>p<0.05 (t-test); Data are mean +/− SD. N = 5.</i></p