Cdc37 interacts with the glycine-rich loop of Hsp90 client kinases

Abstract

Recently, we identified a client-binding site of Cdc37 that is required for its association with protein kinases. Phage display technology and liquid chromatography-tandem mass spectrometry (which identifies a total of 33 proteins) consistently identify a unique sequence, GXFG, as a Cdc37-interacting motif that occurs in the canonical glycine-rich loop (GXGXXG) of protein kinases, regardless of their dependence on Hsp90 or Cdc37. The glycine-rich motif of Raf-1 (GSGSFG) is necessary for its association with Cdc37; nevertheless, the N lobe of Raf-1 (which includes the GSGSFG motif) on its own cannot interact with Cdc37. Chimeric mutants of Cdk2 and Cdk4, which differ sharply in their affinities toward Cdc37, show that their C-terminal portions may determine this difference. In addition, a nonclient kinase, the catalytic subunit of cyclic AMP-dependent protein kinase, interacts with Cdc37 but only when a threonine residue in the activation segment of its C lobe is unphosphorylated. Thus, although a region in the C termini of protein kinases may be crucial for accomplishing and maintaining their interaction with Cdc37, we conclude that the N-terminal glycine-rich loop of protein kinases is essential for physically associating with Cdc37.

FIG. 1.

Cdc37(181-378) interacts directly with the kinase domain of Raf-1. The Myc-tagged kinase domain of Raf-1 and either FLAG-Cdc37 (FL) or FLAG-Cdc37(181-378) (181-378) were expressed in COS7 cells. The cell lysates (whole, lanes 1 and 2) were subjected to immunoprecipitation (IP) with anti-FLAG (IP: α-FLAG, lanes 3 and 4) or anti-Myc (IP: α-Myc, lanes 5 and 6) antibody, followed by immunoblotting (WB) with the indicated antibodies. The asterisk indicates nonspecific bands.

FIG. 2.

The glycine-rich loop of Raf-1 interacts with Cdc37. (A) Schematic of the primary structures of the EGFP-Myc fusion proteins showing the peptide sequences of wild-type (wt) and mutant (ASA) Raf-1; the altered amino acids are marked by a box. M indicates the N-terminal methionine. (B) EGFP-Myc alone (empty) and EGFP-Myc fused to the wild-type or mutant Raf-1 peptide were coexpressed with FLAG-Cdc37(181-378) in COS7 cells. Cell extracts (whole, lanes 1 to 3) were subjected to immunoprecipitation with anti-FLAG (IP: α-FLAG, lanes 4 to 6) or anti-Myc (IP: α-Myc, lanes 7 to 9) antibody, followed by immunoblotting with both anti-FLAG and anti-Myc antibodies. The asterisk indicates nonspecific bands. (C and D) Wild-type and mutant forms of the Myc-tagged Raf-1 kinase domain (Myc-Knd) and either FLAG-Cdc37 (C) or FLAG-Cdc37(181-378) (D) were expressed in COS7 cells. The cell lysates (whole) were subjected to immunoprecipitation with anti-FLAG or anti-Myc antibody, followed by immunoblotting with both anti-FLAG and anti-Myc antibodies. (E) FLAG-Cdc37(181-378) (181-378) was expressed in COS7 cells; the cell lysates (whole, lane 7) and purified proteins (IP, lane 8) were immunoblotted with the indicated antibodies. The purified FLAG-Cdc37(181-378) protein was subjected to SDS-PAGE and Coomassie staining (lane 1). Myc-GST (empty) and wild-type (wt) and mutant (ASA) forms of Myc-GST fused to either the kinase domain of Raf-1 (Knd) or its N lobe were separated by SDS-PAGE and then subjected to either Coomassie staining (lanes 2 to 6) or immunoblotting with the indicated antibodies (lanes 9 to 13). Asterisks indicate unknown proteins that are present in all preparations of the Myc-GST fusion proteins. Molecular mass markers are shown in kDa on the left of the panel. (F) Myc-GST, the Myc-GST-fused kinase domain of Raf-1, and the Myc-GST-fused N lobe of Raf-1, which were adsorbed to glutathione Sepharose, were incubated with FLAG-Cdc37(181-378) and then subjected to a GST pull-down assay. Samples were analyzed by immunoblotting with the indicated antibodies. (G) Wild-type and mutant forms of the His-tagged Raf-1 peptide adsorbed to Ni-NTA agarose were incubated with recombinant GST-Cdc37(181-378) proteins, and then proteins bound to the peptide-adsorbed resin were analyzed by immunoblotting with anti-Cdc37 antibody.

FIG. 3.

Full-length Cdc37 does not bind to the N-lobe or glycine-rich loop of Raf-1. (A) Myc-tagged Raf-1 kinase domain (Knd) and Myc-tagged GST fused to the N-lobe of Raf-1 were coexpressed with either FLAG-Cdc37 or FLAG-Cdc37(181-378) (FL or 181-378, respectively) in COS7 cells (whole, lanes 1 to 4), and the indicated immunoprecipitates (IP: α-FLAG, lanes 5 to 8; IP: α-Myc, lanes 9 to 12) were analyzed by immunoblotting with both anti-FLAG and anti-Myc antibodies. (B) FLAG-Cdc37 or FLAG-Cdc37(181-378) was coexpressed in COS7 cells with EGFP-Myc either alone (e) or fused to the Raf-1 peptide (p) shown in Fig. 2A. Cell extracts were prepared (whole) and subjected to immunoprecipitation with anti-FLAG (IP: α-FLAG) or anti-Myc (IP: α-Myc) antibody, followed by immunoblotting as indicated. Asterisks indicates nonspecific bands.

FIG. 4.

Chimeric mutants of Cdk2 and Cdk4. (A) FLAG-Cdc37(181-378) was coexpressed in COS7 cells with Myc-tagged GST (Myc-GST) either alone (e) or fused to the N lobe of Cdk4 or Cdk2 (4 or 2, respectively) and subjected to immunoprecipitation with anti-FLAG (IP: α-FLAG) or anti-Myc (IP: α-Myc) antibody, followed by immunoblotting with anti-FLAG and anti-Myc antibodies. (B) Schematic of Cdk4, Cdk2, and two chimeric mutants. Numbers in parentheses indicate the total number of amino acids; those in boxes indicate the original residue numbers. (C) FLAG-Cdc37 was coexpressed in COS7 cells with the indicated Myc-tagged kinases; Cdk4, Cdk2, and the two chimeric mutants, Cdk2/4 and Cdk4/2, are indicated as 4, 2, 2/4, and 4/2, respectively. Cell extracts (whole, lanes 1 to 4) were subjected to immunoprecipitation (IP: α-FLAG, lanes 5 to 8; IP: α-Myc, lanes 9 to 12) and immunoblotting with anti-Hsp90, anti-FLAG, and anti-Myc antibodies. (D) Myc-GST fused to the N-terminal portion of the Raf-1 kinase domain (I to IV) and/or the Myc-tagged C-terminal portion of the Raf-1 kinase domain (V to XI) was expressed in COS7 cells in the presence or absence of FLAG-Cdc37 as indicated. Cell extracts (whole) were treated as described for panel C.

FIG. 5.

Unphosphorylated PKA binds to Cdc37. (A) Myc-tagged PKA (Myc-PKA) was coexpressed with FLAG-Cdc37, FLAG-Cdc37(1-180), or FLAG-Cdc37(181-378) in COS7 cells (FL, 1-180, or 181-378, respectively). Cell extracts (whole, lanes 1 to 3) were immunoprecipitated with anti-FLAG (IP: α-FLAG, lanes 4 to 6) or anti-Myc (IP: α-Myc, lanes 7 to 9) antibody and subjected to immunoblotting with both anti-FLAG and anti-Myc antibodies. The two short horizontal lines indicate a doublet band. (B) Myc-PKA and FLAG-Cdc37(181-378) were coexpressed in COS7 cells, and the indicated immunoprecipitates (IP: α-FLAG, lane 1; IP: α-Myc, lane 2) were analyzed by immunoblotting with antiphosphorylated (at Thr¹⁹⁷) PKA (α-pPKA) and anti-Myc (to detect Myc-PKA) antibodies. (C) Myc-PKA was expressed in COS7 cells either alone (control, lane 1 to 3) or with FLAG-Cdc37(181-378) [FLAG-Cdc37(181-378), lanes 4 to 6] and purified by immunoprecipitation with anti-Myc or anti-FLAG antibody, respectively. The purified PKA (input, lanes 1 and 4) was either mock treated (without phosphatase [PPase −], lanes 2 and 5) or treated with phosphatase (PPase +, lanes 3 and 6) and subjected to immunoblotting with anti-Myc antibody. (D) Wild-type and mutant forms of Myc-PKA (wt and TA, respectively) were coexpressed with FLAG-Cdc37 in COS7 cells, and the immunoprecipitates obtained were analyzed by immunoblotting with both anti-FLAG and anti-Myc antibodies. (E) FLAG-tagged PKA was translated from mRNAs with or without a stop codon (+ or −, respectively) in rabbit reticulocyte lysate for 20 min at 30°C. Reaction mixtures were immunoprecipitated with anti-FLAG antibody and subjected to immunoblotting with both anti-phosphorylated PKA and anti-FLAG antibodies. (F and G) After protein synthesis, as described for panel E, PKA released from the ribosome (F, RNase at 0 min) or the ribosome-bound PKA (G, puromycin at 0 min) was incubated for a further 30 min at 30°C in the presence of either 0.2 mg/ml of RNase to terminate translation (F, RNase at 30 min) or 1 mM puromycin to release translation products from the ribosome (G, puromycin at 30 min). Immunoprecipitates obtained with anti-FLAG antibody were subjected to immunoblotting with the indicated antibodies.