Regulation of SR-BI protein levels by phosphorylation of its associated protein, PDZK1

Abstract

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.

Fig. 1.

The C-terminal region of PDZK1 is required for SR-BI up-regulation. (A) Schematic representation of various deletion mutants of PDZK1. (B) Western blot analysis of SR-BI from CHO-K1 cells constitutively expressing various deletion mutants of PDZK1 and transiently transfected with SR-BI. The graph represents relative quantities of SR-BI proteins in these Western blot analyses. (C) Western blot analysis of SR-BI and PDZK1 from cell lysates of Fao cells transduced with the recombinant adenovirus construct of PDZK1. LacZ is a control. The data represent at least three independent experiments.

Fig. 2.

PDZK1 is a phosphorylated protein. (A) Autoradiography (Upper) and the Western blot analysis of PDZK1 (Lower). CHO-KI cells were transiently transfected with an expression plasmid of C-terminal deletion mutants of PDZK1 (Δ457-523, Δ478-523, and Δ500-523) and incubated with [³²P]orthophosphate. (B) Phosphoamino acid analysis of ³²P-labeled PDZK1. Circles indicate where the phosphoamino acid standards migrated. P-Ser, phospho-Ser; P-Thr, phospho-Thr; P-Tyr, phospho-Tyr.

Fig. 3.

Ser-509 of PDZK1 is phosphorylated. (A) Schematic representation of potential sites of Ser phosphorylation in the PDZK1 C-terminal region. (B) Autoradiography (Upper) and the Western blot analysis of PDZK1 (Lower). CHO-KI cells were transiently transfected with various single-point mutants of PDZK1 (S509A, S516A, and S518A) and labeled with [³²P]orthophosphate.

Fig. 4.

PKA phosphorylates PDZK1. (A) Recombinant GST-PDZK1 or GST-S509A mutant fused protein expressed by E. coli was incubated with or without PKA in the presence of [γ-³²P]ATP. (B and C) Metabolic labeling with [³²P]orthophosphate of CHO-K1 cells constitutively expressing PDZK1 were performed in the absence or presence of 10 μM forskolin (B) or 10 μM H-89 (C). Autoradiography (Upper) and CBB staining (Lower) are shown. The data represent at least three independent experiments.

Fig. 5.

PDZK1 expressed in the liver is phosphorylated per se.(A) Preparation of Abs against phospho-Ser-509. The underlined phosphorylated peptide was synthesized for immunization. (B) The recombinant PDZK1 expressed by E. coli was incubated with or without PKA and then subjected to Western blotting using anti-PDZK1 mAb (Left), anti-509-P-PDZK1 pAb (Right). (C) Western blot analysis of PDZK1 from rat liver membrane extract (70 kDa) and the recombinant GST-PDZK1 fused protein (96 kDa) using anti-PDZK1 mAb (Left) and anti-509-P-PDZK1 pAb (Right).

Fig. 6.

SR-BI protein level is regulated by PDZK1 through phosphorylation. Western blot analysis of SR-BI (Upper) and PDZK1 (Lower) from cell lysates of Fao cells transduced with the recombinant adenovirus of PDZK1 or S509A PDZK1. LacZ is a control. The results of this figure are representative of at least three independent experiments.

Fig. 7.

Glucagon increases PDZK1 phosphorylation and hepatic SR-BI expression. Five rats were injected twice daily with 400 μg of glucagon or a vehicle for 2 days. Rats were killed 2 h after the last injection. (A) Total and HDL cholesterol levels. (B) Western blot analysis of SR-BI, PDZK1, phosphorylated PDZK1, and α-actin from rat liver homogenates of two representative animals. (C) Quantitative analysis of SR-BI and PDZK1 transcripts. Total RNA purified from rat liver was subjected to real time RT-PCR for measuring SR-BI and PDZK1 transcripts, as described in Materials and Methods.