Phosphorylation of prion protein at serine 43 induces prion protein conformational change

Abstract

The cause of the conformational change of normal cellular prion protein (PrP) into its disease-associated form is unknown. Posttranslational modifications, such as glycosylation, acetylation, S-nitrosylation, and phosphorylation, are known to induce protein conformational changes. Here, we investigated whether phosphorylation could induce the conformational change of PrP because PrP contains several kinase motifs and has been found recently in the cytosol, in which kinases generally reside. Neuronal cyclin-dependent kinase 5 (Cdk5) phosphorylated recombinant PrP(23-231) at serine 43 (S43) in an in vitro kinase assay. Cdk5-phosphorylated PrP became proteinase K resistant, formed Congo Red-positive fibrils, and formed aggregates that were immunostained with anti-PrP and anti-phospho-PrP(S43) (anti-pPrP(S43)). pPrP(S43) was detected in PrP/Cdk5/p25 cotransfected N2a cells. Roscovitine inhibition of Cdk5 activity or transfection of N2a cells with mutant PrP S43A eliminated the anti-pPrP(S43)-immunopositive protein. Alkaline phosphatase-sensitive and proteinase K-resistant pPrP(S43) immunoreactivity was observed in scrapie-infected but not control-injected mice brains. These results raise the possibility that phosphorylation could represent a physiological mechanism of PrP conversion in vivo.

Figure 1.

Cdk5 phosphorylation of human PrP_23–231 at S43. A, Autoradiogram of Cdk5 in vitro kinase assay on PrP_23–231 or Tau protein. B, Coomassie blue stain of PrP and Tau protein. C, Autoradiogram of in vitro Cdk5 kinase assay on PrP in the absence or presence of olomoucine (Olo.). D, Autoradiogram and Coomassie blue stain of in vitro Cdk5 kinase assay on PrP and PrP S43A. E, Western blot with anti-pPrP^S43 antiserum and anti-PrP^109–112 3F4 antibody of 100 ng of nonphosphorylated PrP or 100 ng of Cdk5 pPrP. F, Western blot with 3F4 antibody of PrP and pPrP immunoprecipitated (IP) with anti-pPrP^S43 or anti-PrP^36–56 R155 antisera. G, Western blot with 3F4 and anti-pPrP^S43 of Cdk5 kinase assay on PrP or PrP S43A.

Figure 2.

Cdk5-phosphorylated, but not CKII-phosphorylated, PrP is resistant to PK. A, Autoradiogram of Cdk5-phosphorylated PrP incubated with 0–10 μg/ml PK for 1 h at 4°C or 37°C. B, Autoradiogram of Cdk5-phosphorylated PrP incubated with 0–50 μg/ml PK for 4 h at 37°C. C, Autoradiogram of Cdk5- or CKII-phosphorylated PrP incubated with 0 or10 μg/ml PK for 1 h at 37°C.

Figure 3.

Cdk5-phosphorylated PrP induces the aggregation of nonphosphorylated PrP in vitro. A, Autoradiogram and Western blot analysis with 3F4 antibody of nonphosphorylated or Cdk5-phosphorylated PrP treated with 10 μg/ml PK for 1 h at 37°C. The arrow indicates the 10 kDa PrP^RES fragment detected on the autoradiogram or Western blot. B, PrP Western blot of nonphosphorylated PrP seeded with kinase assays performed with (pPrP) or without Cdk5 and incubated for the indicated time without (−PK) or with (+PK) PK treatment. The bottom shows a longer exposure of another pPrP seeded experiment revealing the 10 kDa PrP fragment in +PK. C, PrP Western blot of nonphosphorylated PrP seeded with a 2 μl aliquot of the 96 h time point (0 cycle) in B and incubated 24 h (cycle 1). Subsequent cycles represent samples in which 2 μl at the end of the incubation period was added into fresh nonphosphorylated PrP and incubated 24 h. The bottom represents an original seed of 0.2 μl of the 96 h time point in B.

Figure 4.

Cdk5-phosphorylated PrP forms aggregates. A–C, Immunostaining of pPrP with 6H4 (10 nm gold particle size) (A), anti-pPrP^S43 (10 nm) (B), or both anti-pPrP^S43 (18 nm) and 6H4 (5 nm) (C) antibodies. D, Control of fibrillar Aβ_1–42 immunostaining with anti-pPrP^S43 (10 nm). E, F, Immunostaining of nonphosphorylated PrP with 6H4 (10 nm) (E) and anti-pPrP^S43 (10 nm) (F) antibodies.

Figure 5.

Cdk5-phosphorylated PrP forms fibrils. A, Electron micrograph of dialyzed Cdk5-phosphorylated PrP showing fibrillar-like structures. Scale bar, 0.5 μm. B, Fibrils detected in pPrP after a 16 d incubation at 37°C. Scale bar, 0.2 μm for left and right; 2 μm for middle. C, Compact structure of pPrP after a 16 d incubation at 37°C. Scale bar, 2 μm. D, Amorphous appearance of nonphosphorylated PrP after a 16 d incubation at 37°C. Scale bar, 0.5 μm. E, Congo Red staining of dialyzed PrP, Cdk5 alone, Cdk5-pPrP, PrP^RES, fibrillar Aβ_1–42, and Aβ_42–1. Pictures were taken under polarized light microscopy.

Figure 6.

Purification of phosphorylated PrP from PrP/Cdk5/p25 cotransfected N2a cells protein extracts. A–D, Western blot analyses with 3F4, pTyr, anti-pPrP^S43, and β-actin on phospho-column fractionated proteins from PrP/Cdk5/p25 cotransfected N2a cells (A), roscovitine-treated PrP/Cdk5/p25 cotransfected N2a cells (B), PrP S43A/Cdk5/p25 cotransfected N2a cells (C), PrP-transfected N2a (D), or untransfected N2a cells (E).

Figure 7.

Immunohistological staining of control PBS- and 22A scrapie-infected mice brains. Micrographs of control PBS-injected or 22A-infected mice brain tissue sections of the medulla untreated (no primary, anti-pPrP^S43, adsorbed anti-pPrP^S43), pretreated with PK (anti-pPrP^S43 + PK), or pretreated with alkaline phosphatase (anti-pPrP^S43 + AP) with anti-pPrP^S43, no primary antiserum, or adsorbed anti-pPrP^S43.

Figure 8.

Cytosolic and nuclear pPrP^S43 staining of 22A scrapie-infected mice brains. Higher magnification of micrographs of 22A-infected mice brain tissue sections from the thalamus or the medulla immunostained with anti-PrP^S43.