Rho kinase II phosphorylation of the lipoprotein receptor LR11/SORLA alters amyloid-beta production

Abstract

LR11, also known as SorLA, is a mosaic low-density lipoprotein receptor that exerts multiple influences on Alzheimer disease susceptibility. LR11 interacts with the amyloid-β precursor protein (APP) and regulates APP traffic and processing to amyloid-β peptide (Aβ). The functional domains of LR11 suggest that it can act as a cell surface receptor and as an intracellular sorting receptor for trans-Golgi network to endosome traffic. We show that LR11 over-expressed in HEK293 cells is radiolabeled following incubation of cells with [(32)P(i)]orthophosphate. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover putative LR11 interacting kinases. Rho-associated coiled-coil containing protein kinase (ROCK) 2 was identified as a binding partner and a candidate kinase acting on LR11. LR11 and ROCK2 co-immunoprecipitate from post-mortem human brain tissue and drug inhibition of ROCK activity reduces LR11 phosphorylation in vivo. Targeted knockdown of ROCK2 with siRNA decreased LR11 ectodomain shedding while simultaneously increasing intracellular LR11 protein level. Site-directed mutagenesis of serine 2206 in the LR11 cytoplasmic tail reduced LR11 shedding, decreased LR11 phosphorylation in vitro, and abrogated LR11 mediated Aβ reduction. These findings provide direct evidence that LR11 is phosphorylated in vivo and indicate that ROCK2 phosphorylation of LR11 may enhance LR11 mediated processing of APP and amyloid production.

FIGURE 1.

LR11 is phosphorylated in vivo. A, HEK293 cells transfected with empty vector (Vector ctl) or V5-LR11 were metabolically labeled with [³²P_i]orthophosphate for 2 h, as described under “Experimental Procedures.” IPs were performed with V5 antibody and subjected to SDS-PAGE, autoradiography, and immunoblot. The incorporation of ³²P into bands at ∼250 kDa that co-migrate with V5-LR11 immunoreactivity in subsequent immunoblots is consistent with V5-LR11 being a phosphoprotein. The absence of LR11 bands in Vector ctl lane indicates specificity of V5 IP. B, HEK293 cells expressing V5-LR11 were labeled as above and IPs were performed as described under “Experimental Procedures.” Before SDS-PAGE, immunoprecipitates were incubated with calf-intestinal alkaline phosphatase (CIP) or without (Mock) for 1 h at 37 °C. Autoradiography reveals loss of V5-LR11 phosphorylation in CIP-treated samples, and immunoblots indicate that V5-LR11 levels in the IPs are unchanged. Data shown are representative of two independent experiments.

FIGURE 2.

Identification of LR11-ROCK2 interaction by LC-MS/MS. A, flowchart of experimental design to identify LR11-interacting partners. B, representative ROCK2 peptide spectrum (residues 1227–1239) from LR11 IP. C, list of the most abundant candidate LR11-binding partners. Each protein listed was absent from control IP samples. List of all spectra identified in control and V5-LR11 IPs is available in supplemental Table S1. TP, total peptides; TSC, total spectra counts; % Coverage, percent protein coverage.

FIGURE 3.

Biochemical validation of LR11-ROCK2 interaction. A, HEK293 cells transfected with empty vector (Vector ctl) or V5-LR11 were harvested for IP with V5 antibody and proteins were resolved by SDS-PAGE. Immunoblotting revealed the presence of ROCK2 only in V5-LR11 IPs and not in control IPs. B, HEK293 cells were transfected with plasmid expressing V5-LR11 for IPs with mouse Ig plus protein A beads (Ig+beads) or ROCK2 antibody (ROCK2) and subjected to SDS-PAGE. Immunoblotting revealed LR11 only in ROCK2 IPs. Absence of ROCK2 in Ig+beads lane indicates specificity of ROCK2 IP. C and D, reciprocal co-IPs from post-mortem human frontal cortex. C, LR11 IPs using (left to right) protein A beads only (beads), pre-immune sera, or LR11 anti-sera (antisera). Immunoblotting revealed ROCK2 only in antisera IP while the absence of LR11 in control lanes (beads and pre-immune sera) indicate specificity of IP. D, ROCK2 IPs using (left to right) Ig+beads or ROCK2. Immunoblot reveals LR11 only in ROCK2 IP. Input lysate represents 5% by volume of IP. Blots in A and B are representative of three independent experiments. Findings in C and D were replicated in three independent postmortem cases.

FIGURE 4.

Inhibition of Rho kinase reduces LR11 phosphorylation in vivo. A, LR11 IPs from HEK293 cells transfected with V5-LR11 were incubated with kinase buffer-containing cold ATP, [³²P_i]orthophosphate, and DMSO (Mock) or 10 μm Rho kinase inhibitor (RKI) for 30 min at 30 °C, as described under “Experimental Procedures.” Autoradiography indicated that RKI treatment inhibits LR11 phosphorylation in vitro. Immunoblotting identified V5-LR11 and ROCK2 in V5-LR11 IPs. B, HEK293 cells transfected with V5-LR11 were metabolically labeled with [³²P_i]orthophosphate for 2 h in the presence of 50 μm RKI (RKI) or DMSO (Mock). LR11 was IPed with V5 antibody and subjected to SDS-PAGE. Autoradiography revealed that RKI reduced LR11 phosphorylation in vivo. Immunoblot data indicated that relatively equivalent amounts of LR11 were present in each IP. C, intensity of phosphoproteins at 250 kDa was quantified and normalized to the amount of LR11 in the IPs. Data shown in A and B are representative of three independent experiments.

FIGURE 5.

ROCK2 knockdown decreases LR11 ectodomain shedding. A, HEK293 cells were transfected with ROCK2 or Scramble (non-targeting) siRNA smart pools and harvested after 96 h. Media were conditioned for 24 h beginning 72 h after transfection. Equivalent volumes of conditioned media (CM) were resolved by SDS-PAGE. Immunoblot analyses reveal reduction in LR11 ectodomain shedding in ROCK2-depleted cells but no change in APPsα release. For analyses of total cell lysate (Lysate), 50 μg of protein was loaded per lane in SDS-PAGE. Immunoblotting indicated that the reduction in ROCK2 protein was accompanied by an increase in cellular LR11. Calnexin was used as a loading control. B, ROCK2 depletion produced significant changes in cell-associated ROCK2 and LR11 (60% decrease and 33% increase, respectively) and secreted LR11 ectodomain (44% decrease). C, semi-quantitative reverse transcription-PCR analyses demonstrate no change in LR11 mRNA, suggesting that the observed changes in protein levels are not likely to be due to changes in LR11 transcription. β-Actin was used as a control. Data are representative of three independent experiments.

FIGURE 6.

Mutagenesis of LR11 Ser-2206 reduces LR11 ectodomain shedding. A, primary sequence of the cytoplasmic tail of human LR11 indicating in silico predicted sites of ROCK2 phosphorylation and the two serines targeted for mutagenesis. Serine residues at amino acid 2167 and 2206 were substituted for alanines by site-directed mutagenesis, as described under “Experimental Procedures.” B, HEK293 cells were transfected with empty vector (Vector ctl), V5-LR11, V5-LR11_S2167A, or V5-LR11_S2206A and harvested after 48 h. Media were conditioned for 24 h beginning 24 h after transfection. Equivalent volumes of conditioned media (Media) were loaded for SDS-PAGE. Immunoblot analyses indicate ∼46% reduction in LR11 ectodomain shedding in V5-LR11_S2206A samples but no change in V5-LR11_S2167A shedding. For cell lysate (Lysate) analyses, 50 μg of protein was loaded per lane for SDS-PAGE. Immunoblot reveals similar levels of cellular V5-LR11, V5-LR11_S2167A, and V5-LR11_S2206A. For Media and Lysate, a nonspecific immunoreactive band and calnexin, respectively, were used as loading controls. C, intensity of immunoblot bands were quantified and show significant reduction in LR11 ectodomain shedding in LR11_S2206A samples. D, HEK293 cells transfected with empty vector (Vector ctl), V5-LR11 (LR11wt), or V5-LR11_S2206A were harvested for IP with V5 antibody. IPs were incubated with kinase buffer containing cold ATP and [³²P_i]orthophosphate for 30 min at 30 °C. Autoradiography indicates greater intensity of labeled bands in V5-LR11 immunoprecipitates compared with V5-LR11_S2206A, suggesting that Ser-2206 is a potential ROCK2 phosphorylation site in vitro. Immunoblot reveals ROCK2 in V5-LR11 and LR11_S2206A immunoprecipitates, indicating that mutagenesis of LR11 Ser-2206 does not impact LR11-ROCK2 co-IP. Further, immunoblot shows equivalent LR11 enrichment in V5-LR11 and V5-LR11_S2206A IPs. Input lysate represents 5% by volume of IP. Data are representative of three independent experiments. TM, transmembrane domain; wt, wild-type.

FIGURE 7.

LR11 Ser-2206 is necessary for LR11-mediated Aβ reduction. A, HEK293 cells were transfected with empty vector (Vector ctl), V5-LR11, orV5-LR11_S2206A and harvested after 72 h. Media were conditioned for 48 h beginning 24 h after transfection. Equivalent volumes of conditioned media (Media) and 50 μg of protein for cell lysates (Lysate) were loaded for SDS-PAGE. Immunoblot analyses indicate similar levels of secreted APPsα and full-length APP among all samples. Calnexin was used as a loading control. B, endogenous secreted Aβ1–40 was detected by sandwich ELISA. Expression of V5-LR11 reduced secreted Aβ1–40 by 44% while expression of LR11_S2206A reduced secreted Aβ1–40 by only 28% compared with empty vector control. Data shown are representative of three independent experiments. wt, wild-type.