PMC

Phosphorylation of the Thr-143-containing peptide by CK2. Recombinant FKBP52 (amino acids 6–458) (A) or FKBP52 (amino acids 6–148) (B) was phosphorylated by CK2 in the presence of Pept419 or Pept790. The phosphorylation mixtures were analyzed by CBB staining (Upper) or by autoradiography (Lower). (C) Phosphorylation of Pept419 by CK2. Pept419-KLH or Pept790-KLH was phosphorylated in vitro by CK2. CBB staining (lanes 1 and 2) and autoradiography (lanes 3 and 4) are shown. The position of the peptide-conjugates is indicated by an arrow.

Phosphorylation of various deletion mutants of FKBP52 by CK2. (A) Various deletion mutants of FKBP52 were phosphorylated in vitro by CK2. CBB staining is shown in the upper panel and autoradiography in the lower panel. Mutants are designated by their amino acid residue numbers on the top of lanes. The positions of wild-type FKBP52 and molecular mass markers (kDa) are shown on the left. The low molecular mass proteolytic fragments of FKBP52 are indicated by arrowheads. (B) Schematic representation of the phosphorylation of FKBP52 mutants by CK2. Mutants are designated by their amino acid residue numbers, and the degrees of the phosphorylation by CK2 are indicated on the right as follows: −, not at all phosphorylated; +, moderately phosphorylated; ++, strongly phosphorylated; and +++, very strongly phosphorylated.

CK2-phosphorylated FKBP52 does not bind HSP90. (A) Purified recombinant FKBP52 was phosphorylated by CK2 in the presence of [γ-³²P]ATP in vitro. HSP90-binding activity of FKBP52 was examined by native PAGE and CBB staining is shown. Lane I, HSP90 alone (3 μg); lane II, FKBP52 alone (2 μg); or lane III, FKBP52 (2 μg) was mixed with HSP90 (3 μg). The positions of HSP90, FKBP52, and FKBP52–HSP90 complexes are indicated by arrows. Outlines of protein bands are illustrated on the right with the numbers 1–4. (B) Bands 1–4 of A were excised and analyzed by re-electrophoresis on an SDS/polyacrylamide gel. CBB staining (Left) and corresponding autoradiography (Right) are shown. The positions of HSP90 and FKBP52 are indicated by arrows on the left.

Phosphorylation of FKBP52 in vivo. COS7 cells were transfected with an expression plasmid of FKBP52 (amino acids 6–458) and labeled with [³²P]orthophosphate. (A) CBB staining of proteins isolated with FK506–Affi-Gel 10 (lane 2) or with control–Affi-Gel 10 (lane 1). The positions of FKBP52 and HSP90 are indicated by arrows. Two additional associated proteins are indicated by arrowheads. (B) Autoradiography of the same gel as A. In vivo phosphorylated FKBP52 and HSP90 are indicated by arrows. (C) V8 phosphopeptide maps of in vivo- (lane 5) and in vitro- (lane 6) phosphorylated FKBP52 are shown by autoradiography. The position of original FKBP52 is indicated by an arrowhead, and phosphopeptides common to in vivo- and in vitro-phosphorylated FKBP52 are indicated by arrows.

Phosphorylation sites of FKBP52 by CK2. (A) GST (lanes 1 and 4), GST-FKBP52 (amino acids 6–148) (lanes 2 and 5), and GST-FKBP52 (amino acids 32–138) (lanes 3 and 6) were phosphorylated by CK2 in vitro without thrombin cleavage. CBB staining (lanes 1–3) and autoradiography (lanes 4–6) are shown. (B) The amino acid sequence of the corresponding region of rabbit FKBP52. The result in A indicates that the boxed regions should contain the CK2-phosphorylation site(s), whereas the underlined region should not. Nonconsensus serines are indicated by arrowheads, and Thr-143, which agrees well with the CK2-phosphorylatable motif, is in bold type and indicated by an arrow.

Phosphorylation of purified FKBP52 by CK2. Recombinant rabbit FKBP52 expressed in Sf9 cells (lanes 2, 4, 7, 9) or expressed in E. coli (lanes 3, 5, 8, 10) was incubated with (lanes 4, 5, 9, 10) or without (lanes 2, 3, 7, 8) CK2 in the presence of [γ-³²P]ATP and analyzed by SDS/PAGE. Coomassie brilliant blue (CBB) staining (lanes 1–5) and autoradiography (lanes 6–10) are shown. As a control, CK2 was incubated without FKBP52 (lanes 1 and 6). The molecular masses (kDa) of markers are indicated on the left, and the position of FKBP52 is on the right. The positions of proteolytic fragments of FKBP52 are shown by arrowheads.